Depa amen o de Ingenie ía Química. Uni e sidad de Se illa
VALORIZACIÓN DE SUBPRODUCTOS Y RESIDUOS DE LA INDUSTRIA
DEL CANGREJO ROJO EN BASE A SU CONTENIDO PROTEICO
“Valo isa ion o was es and by-p oduc s om Red-C ay ish Indus y
based on hei p o ein con en ”
MANUEL FÉLIX ÁNGEL
Se illa, 30 de no iemb e de 2015
3
Depa amen o de Ingenie ía Química. Uni e sidad de Se illa
TESIS DOCTORAL
VALORIZACIÓN DE SUBPRODUCTOS Y RESIDUOS DE LA INDUSTRIA
DEL CANGREJO ROJO EN BASE A SU CONTENIDO PROTEICO
“Valo isa ion o was es and by-p oduc s om Red-C ay ish Indus y
based on hei p o ein con en ”
Tesis Doc o al p esen ada po D. Manuel Félix Ángel.
Di igida po los Doc o es:
D. An onio F ancisco Gue e o Conejo y D. Albe o Rome o Ga cía
Los Di ec o es El Doc o ando
Fdo. D. An onio Gue e o Conejo Fdo. D. Albe o Rome o Ga cía Fdo. D. Manuel Félix Ángel
Se illa, 30 de no iemb e de 2015
5
D. ANTONIO FRANCISCO GUERRERO CONEJO Y D. ALBERTO ROMERO
GARCÍA, PROFESORES DE LA UNIVERSIDAD DE SEVILLA
INFORMAN:
Que la p esen e Memo ia i ulada “VALORIZACIÓN DE
SUBPRODUCTOS Y RESIDUOS DE LA INDUSTRIA DEL CANGREJO
ROJO EN BASE A SU CONTENIDO PROTEICO” p esen ado po D.
MANUEL FÉLIX ÁNGEL pa a op a al g ado de Doc o con
Mención In e nacional, ha sido ealizada en su mayo ía en el
Depa amen o de Ingenie ía Química de es a Uni e sidad bajo
nues a di ección, po lo que au o izamos su p esen ación.
Y pa a que cons e y en cumplimien o de la legislación igen e,
i mamos el p esen e en Se illa, a 30 de no iemb e de 2015.
D. An onio Gue e o Conejo D. Albe o Rome o Ga cía

7
D . D. Al onso Mazuelos Rojas, Di ec o del Depa amen o de Ingenie ía
Química de la Uni e sidad de Se illa:
CERTIFICA:
Que la Tesis Doc o al que p esen a D. Manuel Félix Ángel ha sido
ealizada den o de la línea “Reología aplicada y ecnología de luidos
complejos”.
9
INDEX
1. ACKNOWLEDGEMENTS. 15
2. SYNOPSIS. 21
3. BACKGROUND 31
3.1. P o eins 33
Amino acids 33
Pep ides and p o eins 35
P o ein s uc u e 35
P ima y s uc u e 36
Seconda y s uc u e 36
Te ia y s uc u e 36
Qua e na y s uc u e 37
P o ein sepa a ion and cha ac e isa ion 38
Dialysis 38
Column ch oma og aphy 39
Ion-exchange ch oma og aphy 39
Size-exclusion ch oma og aphy 39
A ini y ch oma og aphy 40
High-pe o mance liquid ch oma og aphy (HPLC) 40
Elec opho esis 40
P o ein dena u a ion 42
Non-co alen o ces in a p o ein 43
Mechanism o dena u a ion 46
Tempe a u e-induced dena u a ion 46
P essu e-induced dena u a ion 48
Dena u a ion by small-molecula weigh addi i es 49
Dena u a ion induced by pH 50
Techno- unc ional p ope ies o ood p o eins 51
P o ein solubili y 52
E ec o pH 52
Sal concen a ion’s 52
Dena u a ion o p o ein 53
Gela ion 53
The mally i e e sible ( he mose ) gels 53
High P essu e P ocessing (HHP) in ood p ocessing 54
Wa e binding 55
Emulsi ica ion 57
Foaming 58

Acknowledgemen s
17
Como ya se dijo hace muchos años, no sólo de pan i e el homb e.
Así, du an e es os años en los que he ealizado el abajo de esis
doc o al en el Depa amen o de Ingenie ía Química, no sólo he c ecido
p o esionalmen e, sino que además he es ado odeado de una amilia
que me ha acompañado en muchos momen os de mi ida, es po ello
que ha llegado la ho a de endi ibu o a odos ellos y ag adece les
cada ez que han es ado ahí pa a lo que les he necesi ado.
Po ello, quie o ec ea el eco ido que pod ía habe hecho un día
cualquie a a la en ada en el depa amen o.
T as ab i la pue a de en ada del depa amen o, y en ila me en
ese pasillo, que bien pod ía se el de uno de esos hospi ales que an año
llamaban de bene icencia, cabe eco da aquellas eces en las que as
en a Pepe me o ecía é e de o bien si es aba, como él dice,
“condimen ado”, un é mo uno. T as es a pequeña pausa en la que no
sólo había degus ado una bebida con an ás icas p opiedades
an ioxidan es, sino que ambién discu imos sob e la bu oc acia de la
Uni e sidad, me aden o en el depa amen o donde paso po Sec e a ía
donde se encuen a Mon se y sus ayudas con odo los ámi es
adminis a i os (que no ue on pocos y an en aumen o) que enemos
que ealiza o a Manolo y odos sus paseos al Pabellón de B asil,
en egando odos los documen os pa a que es u ie an a iempo.
Finalmen e, como no, ese incón a la izquie da del depa amen o me
ecue da a Felipe y sus mil quehace es con las a eas de di ec o de
depa amen o y An onio y su doble pan alla, que an a u ilidad iene
pa a abaja jun os y e i a la an conocida o ícolis y sus
consiguien es masajes e apéu icos.
T as sali de ese incón del Depa amen o con un ca amelo que
Mon se me ha o ecido, paso po la pue a del labo a o io 3 y no puedo
deja de eco da la salud de la mace a de Ma i Ca men, que con sus
mimos la hace es a esplandecien e, o la son isa de Jenny que se
encuen a hablando con Luisma. Bueno como odo cambia, con Luis,
que aunque pa ezca men i a, Luisma hace ya unos cuan os meses que
se ue y con él el “Re-lio” del depa amen o.
Acknowledgemen s
18
Como no eco da aquellas idas y enidas a ese labo a o io, donde
an as dudas se solucionaban, y donde o as an as su gían como agua
en p oceso de licue acción as el e emo o de lo que somos, química.
La misma que da o ma a las endo inas y oxi ocinas, la misma que nos
pe mi e alcanza la pleni ud como se humano, la misma que nos
pe mi e alcanza aquello conocido como elicidad, la misma que ha
pe mi ido que és a úl ima madu e en una g an amis ad. Sin sabe lo,
comenzamos con 18 años una andadu a jun os, el des ino hizo que
compa ié amos una pa e de nues as idas, y se á el iempo el
enca gado de lab a cada uno de nues os caminos.
En ando ya en el labo a o io 4 pa a imp imi unos a ículos, saludo
a Pablo, que me cuen a las nue as a esu as de su peque, y a Ca los,
so p endiéndome al en a en su despacho con una señal que indica,
¡Pelig o Cangu os! ¿Po qué si aquí no hay de esos? ¡Ahh!, es un
ecue do de cuando es u is e en Aus alia, me quedo mucho más
anquilo. T as sali , Lucía me comen a una de esas a en u as
depo i as que ha i ido el in de semana, siemp e me ha gus ado el
depo e, cie amen e és e no es más que un e lejo de esa ida que
cuando somos pequeños nos espe a ue a de las ins alaciones
depo i as, y como no, nos ayuda a madu a , a c ece . No puedo
ol ida me que después de hace depo e, hay que ecupe a ue zas y
nada mejo que uno de esas cuñas, escas po supues o, que Au o a
nos ae de Los Palacios.
An es de llega al Labo a o io 6, me c uzo con Ana Ma ín que iene
llegando escuchando música, y en la “L” me opo con Juan Ca los, con
él comen o la uina hacia la que se di ige el Be is, ecue do aquellos
ma a ones de p ác icas, dónde eníamos nues o descanso pa a
come nos nues o abanico, ibé ico po supues o, y comen a el
ascu so de esos pequeños momen os que cons uyen la ida.
P egun o po Paco, pe o es á en la Escuela, allí donde se pasa an as y
an as ho as, y donde ambién hemos compa ido algunas ho as en ese
labo a o io de la edad mode na. Finalmen e, Nie es me da con la mejo
de las son isas el ma az de ondo edondo que le pedí el día an e io ,
pe o mi ando bien, hay alguien más, Na i, que además de adecen a
odo aquello po donde pasa, an as isas gas a en es e luga llamado
“L”.
Acknowledgemen s
19
T as sali , me encuen o con José An onio, que iene de su
labo a o io 5 y me comen a sus úl imos log os en el Ba le ield, su
ines abilidad en el pH del agua de la pece a o los úl imos a añazos que
le ha p opiciado su ga a. Nos di igimos a isi a a Paco, a hace le una de
esas p egun as que no pueden se o a cosa más que ocu encia de José
An onio. Paco in en a comp ende la sin pone ca a de “yo no en iendo
nada”, as lo que no puede hace más que ei . Nu ia nos escucha, y
me comen a que es e in de semana a a pasa se po La Palma, MI
PUEBLO!! ¡Cuidado! po allí hay gen e pelig osa, le ad ie o. De camino
al labo a o io 6, saludo a Mc. Al a o y paso po el despacho de Cecilio,
bueno despacho o con esiona io, po que c eo que lo que ese despacho
no haya oído…, po que CEcilio siemp e iene 5 minu os pa a escucha a
cualquie a, pa a soluciona cualquie duda, pa a hace aquello con lo
que si odos hicié amos el Mundo i ía mejo : escucha y se amable,
ol idando odos aquellos p ejuicios, o gullos y ecelos que nos p ohíben
se elices.
T as odas las pa adas en odos esos incones que o ma el
depa amen o, se a haciendo a de, así que ya engo que en a en el
labo a o io 6, donde es án Albe o y Víc o . Víc o es á e minando uno
de esos abajos del Más e que an a “emoción” gene a, y Albe o
p epa ándose una de esas clases que luego an o éxi o iene en e sus
alumnos, una de esas clases en las que alumnos suyos, que a la ez son
conocidos míos, me pa an y me comen an su magní ica esceni icación.
Ha llegado la ho a de abaja , con la ilusión de hace las cosas bien, sin
ene miedo al u u o, a sabiendas que as la de ensa del abajo de
an os años se ce a á una e apa, pe o con la ilusión que as el cie e
de una pue a se ab e un en anal.
Du an e es os años de esis, ambién han o mado pa e de mí mis
amigos y aquellas pe sonas con las que he i ido, Dani, Ma y, Ja i, Moi,
Juanjo, Jo ge e Inma, así como con odos aquellos con los que he
omado una ce eza y he amenizado los ines de semana en la Palma,
Ismael, Juandi, Juan, Jaime, an, así como de o os an o que han
es ado conmigo en las du as y en las madu as. Aquellas pe sonas que
po sue e o aza del des ino he conocido en los o os luga es donde he
lle ado a cabo mi labo in es igado a, a Heline y Ra en Bélgica, a Tu id,
Acknowledgemen s
20
T ude, Si i, Gianca los en No uega o a Ad i, Espe, Isa, O ega, Rocío o
Inma en Huel a.
Po úl imo, no puedo ol ida me de mi amilia, aquellos que han
o mado pa e de mi ida desde que nací (pues o que soy el he mano
pequeño), aquellos que me han is o aleg e y en adado, aquellos que
me han guiado a lo la go de an os años, aquellos que sé que nunca me
abandona án y siemp e me que án, aquellos a los que dedica é unas
líneas más adelan e. No puedo ol ida me de aquellas pe sonas que ya
no o man pa e de aquello que conocemos como amilia, bien po que
ya no es án en e noso os, como mis abuelos, bien po que
ac ualmen e no lo sean, pe o lo se án. Aquellos en los que end á
in luencia es e p esen e, que pa a en onces se á pasado, aquellos en
los que in lui é e in lui án en un u u o. A odos us edes os engo en
men e en es e p eciso momen o.
2. Synopsis.

Synopsis
23
E e y yea ood and ag icul u al indus y p oduce a big amoun o
su pluses and was es which a e disca ded o used as a low added alue by-
p oduc . A pa icula ly ele an example o his ac is loca ed in Andalusia
(sou he n Spain) and i is associa ed o he ed-swamp c ay ish. This
c us acean was in oduced in he middle o he wen ie h cen u y and due o
a ou able wea he condi ions, abundan ood and he lack o p eda o s, i
has unde gone a as widesp ead g ow h (Ki ja ainen and Wes man 1999).
This apid g ow h has con ibu ed o he de elopmen o a s ong local
c ay ish indus y a he ma shes o he Guadalqui i Ri e (Geige , Alco lo e
al. 2005). A cu en ly a ac i e way o alo ise hese p oduc s is aking
bene i s om i s ela i ely high p o ein con en .
C us aceans cons i u e an excellen sou ce o high-quali y p o ein, ich
no only in essen ial amino acids and lipids, including long-chain
polyunsa u a ed a y acids om ω-3, bu also con aining o he componen s
o unc ional alue such as as axan hin ha possess a high an ioxidan
capaci y, e en highe han o he s impo an an ioxidan s such as β-ca o ene
o i amin E (Miki 1991). The quali y o his p o ein concen a e will con ibu e
o inc ease he unc ional p ope ies in hei p oduc s and de i a i es.
To ob ain a use ul c ay ish p o ein concen a e o hese h ee
applica ions, a p e ious s age o cha ac e iza ion and op imiza ion o di e en
lou s om c ay ish pulp has been ca ied ou . This s age was de eloped by
PEVESA, who selec ed he empe a u e, pH, ex ac ion p ocedu e and p o ein-
dispe sion ac ion in o de o ob ain he phase wi h he highes con en o
soluble p o ein.
This s udy is ocused on di e en applica ions o c ay ish p o ein sys ems
de i ed om hei unc ional p ope ies, aiming a he achie emen o high
alue-added c ay ish-based p oduc s. The applica ions conside ed in his
Synopsis
24
s udy will be: emulsi ica ion abili y, gel o ma ion and bioplas ics p ocessing,
and.
Fi s o all, one applica ion come om ood indus y, which is eally
in e es ed in p oduce s able oil-in-wa e (o/w) emulsions con aining a p o ein
di e en o egg-yolk p o ein as he only emulsi ie in o de o p oduce ood
p oduc s such as mayonnaise and salad d essings. These al e na i e p o eins
would a oid he p esence o choles e ol om yolk, he de elopmen o
salmonella in yolk-con aining ood p oduc s o alle gic eac ions, which
nowadays a e mo e and mo e equen . O he au ho s ha e used p e iously
myo ib illa p o eins such as ac omyosin o p oduce emulsions and ha e
demons a ed ha ac omyosin om hake had highe emulsi ying ac i i y and
s abili y han he ac omyosin om chicken and po k (Co ades, Ca ballo e al.
1996). P o ein om c ay ish may cons i u e an excellen sou ce o p o ein
which may con ibu e o s abilize o/w emulsions.
In addi ion, o he unc ional p ope y is he abili y o p o eins o dena u e
o m agg ega es and adop gel-s uc u e a e a con olled hea ing. This
applica ion con ibu es o design a wide a ie y o ood sys ems wi h he
desi e heological consis ency, mic os uc u e and ex u e. C us acean mea
con en s mainly sa coplasma ic and myo ib illa p o eins. Sa coplasmic
ac ion wi h a globula and ela i ely simple s uc u e, show a weak gela ion
capaci y and, he e o e, a li le con ibu ion o he ex u e o p ocessed oods.
On he o he hand, myo ib illa p o eins, specially myosin and ac omyosin,
cons i u e mul iples dominions ha end o o m iscoelas ic ne wo ks and
gels wi h high consis ency (Damoda an 1997). Finally, an in e es ing a ea o
esea ch is he an ioxidan cha ac e isa ion o hese gels. Due o he p esence
o he abo e men ioned unc ional ing edien s, hese gels may cons i u e an
excellen sou ce o p o ein o he human nu i ion.
Synopsis
25
Finally, a cu en ly a ac i e way o alo ise hese by-p oduc s is h ough
he use o his p o ein concen a e as enewable esou ces in he manu ac u e
o “g een ma e ials”, eplacing ha dly deg adable plas ic ma e ials om oil-
based syn he ic polyme s. Recen ly, some impo an applica ions o
bioplas ics a e beginning o eme ge in he a eas o ood-packaging,
pha maceu ics, elec onics, au omo i e indus y and biomedicine. Thus,
among o he applica ions, bioplas ics om p o eins can be used in ood
packaging, ui coa ing, encapsula ion, ex iles, abso ben ma e ials o issue
enginee ing (Sha ma and Luzino 2012, So oudi and Jakubowicz 2013). This
wide a ie y o po en ial applica ions allows us o en isage an inc easingly use
o biobased-plas ic ma e ials in a nea u u e.
Thus, one o he main objec i es o his s udy has been o e alua e he
po en ials o c ay ish concen a e as emulsi ie o ob ain highly concen a ed
oil/wa e emulsions such as mayonnaise-like. Fi s o all, i was necessa y o
op imize he p ocessing and composi ion pa ame e s ha would lead o long-
e m s able emulsions. In his con ex , in e acial measu emen s we e
pe o med in o de o acili a e p edic ion o emulsion s abili y. Rele an
ul ima e in o ma ion on emulsion s abili y was ob ained by cha ac e izing he
heological p ope ies and mic os uc u e o c ay ish-based emulsions. The
heological cha ac e iza ion has been ocused on he linea iscoelas ic
p ope ies o he emulsions, de e mined by means o small-ampli ude
oscilla o y shea (SAOS). Mic os uc u al pa ame e s ha e been e alua ed
h ough d ople size dis ibu ions analysis. Mic os uc u e has also been
cha ac e ised by Con ocal Lase Scanning Mic oscopy (CLSM).
Ano he adi ional objec i e has been o e alua e he gel abili y and he
bioac i e po en ial o gels made om non-dena u ed c ay ish p o ein
concen a e a di e en pH alues. To explo e imp o emen s in bo h, he
gela ion and bioac i e abili y abili y, di e en hyd olysa es we e ob ained

Backg ound. P o eins
33
3.1. P o eins
P o eins a e he in e media e in e e y p ocess ha akes place in a li ing
being. P o eins also a e he mos abundan biological mac omolecules, being
conside ed e en as biopolyme s.
All p o eins a e buil om he same se o 20 amino acids which a e
co alen ly linked in a cha ac e is ic linea sequence. Each ype o p o ein has
a unique sequence o amino acids which p o ides each o hem wi h
dis inc i e chemical p ope ies. This g oup o 20 p ecu so molecules may be
conside ed as he alphabe used o w i e he p o ein s uc u e and
unc ionali y. P o eins a e ound in a wide ange o sizes, om ela i ely small
pep ides o huge biopolyme wi h molecula weigh o e he millions
(Lehninge , Nelson e al. 2005).
Amino acids
All o he 20 amino acid a e α-amino acids. Each o hem ha e a ca boxyl
g oup and an amino g oup bonded o he same ca bon a om ( he α-ca bon),
and an “R” g oup. They di e om each o he in hei “R” g oups, which a y
in s uc u e, size, and elec ic cha ge. Fo all he amino acids excep o he
glycine, he α-ca bon is bonded o ou di e en g oups: a ca boxyl g oup, an
amino g oup, he “R” g oup, and a hyd ogen a om. Acco ding o his s uc u e,
he α-ca bon a om is a chi al cen e. Thus, he ou di e en g oups can
occupy wo unique spa ial a angemen s due o he e ahed al a angemen
o he bonding o bi als a ound he α-ca bon a om, such ha each amino acid
ha e wo possible s e eoisome L and D. Howe e , all he amino acids
syn he ized by li ing being a e L-amino acid (Alu u 2005). Figu e 3.1-1 shows
he 20 amino acids classi ied by “R” g oup na u e (non-pola , pola -uncha ged,
pola -posi i ely cha ged, pola -nega i ely cha ged and a oma ic).
Backg ound. P o eins
34
Amino acids in aqueous solu ion also can ac as acids and bases. The
amino and ca boxyl g oups o amino acids, along wi h he ionisable R g oups
o some amino acids, can wo k as weak acids and bases. When an amino acid
loss an ionisable R g oup and is dissol ed in wa e a neu al pH, i exis s in
solu ion as he dipola ion, o zwi e ion which can ac as ei he an acid o a
base. These kind o subs ances which ha e an ampho e ic beha iou a e
named ampholy es (Lehninge , Nelson e al. 2005). The wen y amino acids
a e: Glycine, (Gly); His idine, (His); Isoleucine, (Ile); Leucine, (Leu); Lysine,
(Lys); Me hionine, (Me ); Phenylalanine, (Phe); P oline, (P o); Se ine, (Se );
Th eonine, (Th ); T yp ophan, (T p); Ty osine, (Ty ); Valine, (Val).
Figu e 3.1-1: Chemical composi ion and s uc u e o he wen y amino acids,
classi ied by “R” g oup na u e
Backg ound. P o eins
35
Pep ides and p o eins
Pep ides and p o eins a e he polyme s o amino acids. Two amino acid
molecules can be co alen ly bounded h ough an amide linkage, leading a
dipep ide. Such a linkage is o med by emo al o one hyd oxyl-g oup om
he ca boxyl g oup o he s a ing amino acid and one hyd ogen om he
amino g oup o he ollowed amino acid. As a consequence, o ma ion o a
pep ide bond is a condensa ion eac ion whe e each pep ide joining elease a
molecule o wa e . Figu e 3.1-2 schema ises his chemical eac ion.
Figu e 3.1-2: Fo ma ion o pep ide bond om wo amino acids
Th ee amino acids can be joined by wo pep ide bonds o o m a
ipep ide. In he same way, amino acids can be joined o o m e apep ides,
pen apep ides, e c. When a ew amino acids a e linked in his way, he
s uc u e is called an oligopep ide ( om 6 o c.a. 20 amino acids). When many
amino acids a e joined, he p oduc is called a polypep ide. P o eins ha e
housands o amino acid. In ac , polypep ides whose molecula weigh is
highe han 10.000 Da, a e gene ally called p o eins (Lehninge , Nelson e al.
2005).
P o ein s uc u e
Fo la ge mac omolecules such as p o eins, he ask o desc ibing and
unde s anding s uc u e is app oached a se e al le els o complexi y,
a anged in a kind o concep ual hie a chy. Fo p o eins, ou le els o p o ein
s uc u e a e commonly de ine (Alu u 2005).
Backg ound. P o eins
36
P ima y s uc u e
The mos impo an elemen o he p ima y s uc u e is he sequence o
amino acid esidues. The di e ences in p ima y s uc u e can be especially
in o ma i e. Each p o ein has a dis inc i e numbe and sequence o amino
acid esidue. The unc ion o a p o ein depends on i s amino acid sequence.
Seconda y s uc u e
Seconda y s uc u e is e e ed o pa icula ly s able a angemen s o
amino acid esidues gi ing ise o ecu ing s uc u al pa e ns. Thus, he e m
seconda y s uc u e e e s o any chosen segmen o a polypep ide chain,
desc ibing he local spa ial a angemen o i s main-chain a oms, wi hou
ega d o i s ela ionship o o he segmen s.
A egula seconda y s uc u e occu s when each dihed al angle (φ and ψ)
emains he same. The mos commonly s uc u e a e α-helix and β-
con o ma ions. Whe e a egula pa e n is no ound, he seconda y s uc u e
is some imes e e ed o a andom coil Howe e , his con o ma ion does no
desc ibe p ope ly a s uc u e.
Te ia y s uc u e
Te ia y s uc u e desc ibes all aspec s o he h ee-dimensional olding
o a polypep ide. Thus, he o e all h ee-dimensional a angemen o all
a oms in a p o ein is e e ed o as he p o ein's e ia y s uc u e. Amino acids
ha a e a apa in he polypep ide sequence and a e in di e en ypes o
seconda y s uc u e may in e ac wi hin he comple ely olded s uc u e o a
p o ein. The loca ion o bends in he polypep ide chain and he di ec ion and
angle o hese bends a e de e mined by he numbe and he loca ion o
speci ic bend-p oducing con o ma ion. Di e en In e ac ing segmen s o
polypep ide chains a e held in hei cha ac e is ic e ia y loca ion, such as
Backg ound. P o eins
37
se e al kinds o weak in e ac ions o e en by co alen bonds (e.g. disulphide
bonds) be ween di e en p o ein segmen s.
Qua e na y s uc u e
When a p o ein has wo o mo e polypep ide subuni s, hei a angemen
in space is e e ed o as qua e na y s uc u e. Thus, he a angemen o
p o eins which con ain wo o mo e sepa a e polypep ide chains, o subuni s,
in h ee-dimensional complexes cons i u es he qua e na y s uc u e.
Conside ing hese highe le els o s uc u e, i is e y use ul o classi y p o eins
in o wo main g oups: ib ous p o eins (whose polypep ide chains a e
a anged in long s ands o shee s) and globula p o eins (whose polypep ide
chains a e olded in o a sphe ical o globula shape) (Lehninge , Nelson e al.
2005).
Figu e 3.1-3 illus a e he ou di e en con o ma ions which a e adop ed
by a p o ein:
Figu e 3.1-3: Con o ma ions adop ed by a p o ein.

Backg ound. P o eins
38
P o ein sepa a ion and cha ac e isa ion
To s udy a p o ein in de ail, he esea che mus be able o sepa a e i
om o he p o eins in pu e o m and mus ha e he echniques o de e mine
i s p ope ies which allow o unde s and i s beha iou and i s ela ionship wi h
o he mac o and mic o molecula sys ems.
Thus, in any p o ein pu i ica ion, he i s s ep is he p o eins
solubilisa ion in o a solu ion, which is called a c ude ex ac , is he sepa a ion.
Se e al me hods a e a ailable o sepa a ing one o mo e o he p o eins om
he c ude ex ac . Usually, he ex ac is subjec ed o ea men s ha sepa a e
he p o eins in o di e en ac ions based on a physical p ope y such as size
o cha ge. A sepa a ion based on p o ein solubili y (which depends on pH,
empe a u e and sal concen a ion, among o he ac o s) is commonly used
in an ea ly ac iona ion s ep. Fo ins ance, he solubili y o p o eins is
gene ally lowe ed a high sal concen a ions. Thus, he addi ion o ce ain
sal s in he igh amoun can selec i ely p ecipi a e some p o eins, while
o he s will emain in solu ion (Lehninge , Nelson e al. 2005).
Dialysis
A solu ion con aining he p o ein o in e es mus usually be modi ied
be o e he subsequen pu i ica ion s ep. Dialysis is a p ocedu e ha sepa a es
p o eins om small solu es because o he la ge size o p o eins. The pa ially
pu i ied ex ac is placed in a bag o ube made o a semipe meable
memb ane. When his is suspended in a much la ge olume o bu e ed
solu ion o app op ia e ionic s eng h, he memb ane allows he exchange o
sal and bu e bu no o p o eins. Thus, dialysis e ains la ge p o eins wi hin
he memb anes, allowing he concen a ion o o he solu es in he p o ein
p epa a ion o change un il hey come in o equilib ium wi h he solu ion
ou side he memb ane.
Backg ound. P o eins
39
Column ch oma og aphy
The mos powe ul me hods o ac iona ing p o eins make use o
column ch oma og aphy echnique, whe e a bu e ed solu ion ( he mobile
phase) pe cola es h ough i . The p o ein-con aining solu ion, laye ed on he
op o he column, pe cola es h ough he solid ma ix. Indi idual p o eins
mig a e as e o mo e slowly h ough he column depending on hei physical
p ope ies.
Ion-exchange ch oma og aphy
This echnique makes use o di e ences in he sign and magni ude o he
ne elec ic cha ge o p o eins a a selec ed pH. The column ma ix is a
syn he ic polyme con aining bound cha ged g oups. I hose g oups a e
bound wi h anionic g oups om p o ein a e, he column will be called ca ion
exchange s column, and hose wi h bound ca ionic g oups a e called anion
exchange s. The a ini y o each p o ein o he cha ged g oups on he column
is in luenced by he pH (which de e mines he ioniza ion s a e o he molecule)
and he concen a ion o ee sal ions in he su ounding solu ion.
Sepa a ion can be op imized by g adually changing he pH and/o sal
concen a ion o he mobile phase so as o c ea e a pH o sal g adien .
Size-exclusion ch oma og aphy
This echnique is also called gel il a ion because i sepa a e p o eins
acco ding o size. In his me hod, la ge p o eins eme ge om he column
soone han small ones. The solid phase consis s o c oss-linked-polyme
beads wi h enginee ed po es o ca i ies o a pa icula size. La ge p o eins
canno en e he ca i ies and ake a sho (and apid) pa h h ough he
column, a ound he beads. By he con a y, small p o eins en e he ca i ies
and a e slowed by he longe pa h h ough he column.
Backg ound. P o eins
40
A ini y ch oma og aphy
This echnique is based on binding a ini y. The beads in he column ha e
a co alen ly a ached chemical g oup called ligand (a g oup o molecule ha
binds o a mac omolecule such as a p o ein). When a p o ein mix u e is added
o he column, any p o ein wi h a ini y o his ligand binds o he beads, and
i s mig a ion h ough he ma ix is e a ded.
High-pe o mance liquid ch oma og aphy (HPLC)
HPLC makes use o high-p essu e pumps ha speed he mo emen o he
p o ein molecules down he column, as well as highe -quali y
ch oma og aphic ma e ials ha can wi hs and he c ushing o ce o he
p essu ized low. By educing he ansi ime on he column, HPLC can limi
di usional sp eading o p o ein bands and hus g ea ly imp o e esolu ion
(Lehninge , Nelson e al. 2005).
Elec opho esis
Elec opho esis is one o he mos commonly echnique o he
sepa a ion o p o eins based on he mig a ion o cha ged p o eins in an
elec ic ield.
This p ocedu e is no gene ally used o pu i y p o eins in la ge amoun s,
because usually a e a ailable simple al e na i es han elec opho e ic
me hods, which o en a ec he s uc u e and, as a consequence he unc ion
o p o eins. Elec opho esis is howe e especially use ul as an analy ical
me hod. I s ad an age is ha p o eins can be isualized as well as sepa a ed,
pe mi ing a esea che o es ima e quickly he numbe o di e en p o eins
in a mix u e o he deg ee o pu i y o a pa icula p o ein p epa a ion. Also,
elec opho esis allows de e mina ion o c ucial p ope ies o a p o ein such as
i s isoelec ic poin and app oxima e molecula weigh .
Backg ound. P o eins
41
Elec opho esis o p o eins is gene ally ca ied ou in gels made up o he
c oss-linked polyme polyac ylamide (PAGE). The polyac ylamide gel ac s as a
molecula sie e, slowing he mig a ion o p o eins app oxima ely in
p opo ion o hei cha ge- o-mass a io. Mig a ion may also be a ec ed by
p o ein shape. In elec opho esis, he o ce mo ing he mac omolecule is he
elec ical po en ial, E. The elec opho e ic mobili y, µ, o a molecule is he
a io o i s eloci y, V, o he elec ical po en ial. Elec opho e ic mobili y is
also equal o he ne cha ge, Z, o he molecule di ided by he ic ional
coe icien , , which e lec s in pa he p o ein's shape. Thus:
𝜇=𝑉
𝐸=𝑍
𝑓
(3.1-1)
Thus, as can be deduced om Equa ion 3.1-1 (3.1-1) he mig a ion o a
p o ein in a gel du ing elec opho esis may be ega ded as a unc ion o i s size
and shape.
An elec opho e ic me hod commonly employed o es ima ion o pu i y
and molecula weigh makes use o he su ac an sodium dodecyl sulpha e
(SDS), hen he elec opho esis is called SDS-PAGE. SDS binds o mos p o eins
in amoun s oughly p opo ional o he molecula weigh o he p o ein, abou
one molecule o SDS o e e y wo amino acid esidues. The bounded SDS
con ibu es a la ge ne nega i e cha ge, ende ing he in insic cha ge o he
p o ein insigni ican and con e ing on each p o ein a simila cha ge- o-mass
a io. In addi ion, SDS binding pa ially un olds p o eins, such ha mos SDS-
bounded p o eins assume a simila shape. The e o e, elec opho esis in he
p esence o SDS sepa a es p o eins almos exclusi ely on he basis o mass
(molecula weigh ), wi h smalle polypep ides mig a ing as e . A e
elec opho esis, he p o eins a e isualized by adding a dye, such as
Coomassie blue, which binds o p o eins bu no o he gel i sel . The
molecula weigh o each p o ein ac ions is ob ained by compa ison wi h
s anda d p o eins whose molecula weigh is known. I he p o ein has wo o
Backg ound. P o eins
48
In addi ion, se e al p o eins ha e been shown o unde go dena u a ion
a cold empe a u es. The cold empe a u e-induced dena u a ion o p o eins
is mainly due o a dec ease in he s abili y o hyd ophobic in e ac ions a low
empe a u es. The ac ha he hyd ophobic and he con o ma ional en opy
a e he wo ele an o ces, con olling he modynamic s abili y o p o eins
en a i ely sugges s ha he s abili y o p o eins migh be in some way
dependen on he amino acid composi ion.
Finally, ac o s such as wa e con en o d y p o ein powde s o he
p esence o small-molecula -weigh subs ances a ec he mal p o ein
dena u a ion. Thus, as he wa e con en is inc eased, he dena u a ion
empe a u e o p o eins dec eases asymp o ically owa ds a alue ha is
simila o he dena u a ion empe a u e o he p o ein in a dilu e solu ion. This
is due o he plas icizing e ec o wa e , which p omo es segmen al mobili y
in p o eins. On he o he hand, small-molecula -weigh solu es, such as sal s
and suga s, gene ally leads o an inc ease in he dena u a ion empe a u e o
p o eins.
P essu e-induced dena u a ion
P o eins a e inhe en ly highly lexible. This high lexibili y is he
unde lying eason o hei ma ginal s abili y unde physiological condi ions.
The lexibili y o p o eins a ises because o un illed spaces o ca i ies in he
in e io o he p o ein. These ca i ies a e c ea ed by impe ec packing o he
esidues as he p o ein chain collapses on i sel du ing olding, he pa ial
speci ic olume (𝜈0), o a p o ein consis s o EQ. (3.1-3):
𝜈0=𝑣𝑐+𝑣𝑐𝑎𝑣+∆𝑣𝑠𝑜𝑙
(3.1-3)
whe e c is he sum o cons i u i e speci ic olumes o a oms in he
p o ein, Δ sol is he speci ic olume o ca i ies in he p o ein, and and Δ sol is
he speci ic olume change because o he hyd a ion p ocess. The i s e m is

Backg ound. P o eins
49
cons an o a gi en p o ein, while he las wo e ms a e he main pa ame e s
a ec ing he speci ic olume o p o eins.
Unde e y high hyd os a ic p essu e, he collapse o he ca i ies o med
as a esul o impe ec packing o amino acid esidues causes un olding o he
p o ein. In he un olded s a e, elimina ion o he ca i ies dec eases he
olume, and hyd a ion o he exposed hyd ophobic esidues also leads o a
educ ion in he olume o he sol en . Despi e, p essu e-induced gels a e
so e in ex u e han hea -induced gels, and ea u e he capabili y o e aining
colou , la ou , i amins and o he nu ien s ha a e des oyed o some
ex en in he mally p ocessed oods and in hea -induced gels (Damoda an
1997).
Dena u a ion by small-molecula weigh
addi i es
Se e al small-molecula -weigh solu es, such as u ea, guanidine
hyd ochlo ide, su ac an s, suga s, and neu al sal s, a ec p o ein s abili y in
aqueous solu ions. While u ea, guanidine hyd ochlo ide, and small-molecula -
weigh su ac an s des abilize he na i e con o ma ion o p o eins, suga s
end o s abilize he na i e s uc u e. As o neu al sal s, while ce ain sal s
e med as kosmo opes end o s abilize p o ein s uc u e (e.g. sulpha e and
luo ide sal s o sodium), o he sal s, such as b omide, iodide, pe chlo a e, and
hiocyana e, e med as kosmo opes, des abilize p o ein s uc u e.
The s abilizing o des abilizing e ec s o small molecula -weigh addi i es
on p o eins is belie ed o ollow a gene al mechanism. This is ela ed o hei
p e e en ial in e ac ion wi h he aqueous phase and he p o ein su ace.
Addi i es ha s abilize p o ein s uc u e bind e y weakly o he p o ein
su ace bu enhance p e e en ial hyd a ion o he p o ein su ace. Such
addi i es a e gene ally excluded om he egion su ounding he p o ein and,
Backg ound. P o eins
50
as a esul , hei concen a ion nea he p o ein is lowe han in he bulk
solu ion. This concen a ion g adien p esumably c ea es an osmo ic p essu e
g adien su ounding he p o ein molecule, su icien enough o ele a e he
he mal dena u a ion empe a u e.
In he case o addi i es ha des abilize p o ein s uc u e, he opposi e
seems o be ue. Tha is, hose addi i es ha dec ease he s abili y o p o eins
p e e en ially bind o he p o ein su ace and cause dehyd a ion o he
p o ein. In such cases, wa e molecules a e excluded om he egion
su ounding he p o ein and he concen a ion o he addi i e in his wa e -
excluded egion is highe han in he bulk sol en . Fa ou able in e ac ions o
such addi i es wi h p o ein su aces, pa icula ly he non-pola su aces,
p omo e un olding o he p o ein such ha he bu ied nonpola su aces a e
u he exposed o a ou able in e ac ions wi h chao ope addi i es.
Anionic su ac an s such as sodium dodecyl sulpha e (SDS), a e po en
dena u ing agen s wi h he skill o de eloping s ong binding o hyd ophobic
g oups in he c e ices o p o ein molecules, which leads o des abiliza ion and
solubilisa ion o bu ied hyd ophobic egions. Because o his high binding
capaci y, p o eins in SDS solu ion become highly nega i ely cha ged. The
esul an elec os a ic epulsions be ween segmen s also play a ole in p o ein
un olding as well as in p e en ing p o ein agg ega ion.
Dena u a ion induced by pH
Wi h ega ds o pH-induced dena u a ion, p o eins a e ei he nega i ely
o posi i ely cha ged a neu al pH. Na i e p o eins, a his physiological pH,
p esen an equilib ium s uc u e wi h a global minimum ee ene gy ha has
al eady aken in o accoun he p e-exis ing epulsi e and a ac i e
elec os a ic in e ac ions. Howe e , a pH alues a away om he neu al
pH, changes in he s a e o ioniza ion o a ious cha ged esidues in p o eins
al e he elec os a ic ee ene gy, esul ing in con o ma ional changes. Mos
Backg ound. P o eins
51
p o eins a e e y s able a hei isoelec ic poin (IEP), pH a which he ne
cha ge o he p o ein is ze o and elec os a ic epulsi e in e ac ions a e a a
minimum. Howe e , p o eins ypically un old a pH alues a om he IEP,
ha is, below 4 and abo e 10.
This un olding is no simply because o changes in he ioniza ion s a e o
he cha ged esidues on he su ace o he p o ein, bu is ela ed o ioniza ion
o esidues ha a e pa ially o ully bu ied in he p o ein (Hui 2006).
Techno- unc ional p ope ies o ood
p o eins
Func ionali y has been desc ibed as he se o non-nu i i e oles ha
ood cons i uen s play in a ood sys em. Mo e o mally, echno- unc ional
p ope ies a e he physical and chemical p ope ies ha a ec he beha iou
o molecula cons i uen s in ood sys ems.
P o eins in oods a e mul i unc ional and may be he p incipal s uc u al
componen in many ood sys ems, including p oduc s om mea and poul y,
eggs, dai y, ce eals, and legumes. In ac , p o eins con ibu e signi ican ly o
he senso y a ibu es and o e all quali y o ood p oduc s. In his sense,
p o ein unc ionali y is conside ed c i ical o he imp o emen o exis ing
ood p oduc s o he de elopmen o new ones. An example is he use o less
expensi e p o ein sou ces as eplacemen s in adi ional ood p oduc s. Use
o less expensi e p o eins no only allows o cos educ ion, bu also can
inc ease he u iliza ion o ood ma e ials ha p e iously migh ha e been
conside ed was e p oduc s.
Techno- unc ional p ope ies commonly associa ed wi h p o eins include
solubili y, gela ion, emulsi ica ion, oaming, and wa e -holding capaci y
(Damoda an 1997, Alu u 2005).
Backg ound. P o eins
52
P o ein solubili y
The solubili y o a p o ein, which is de e mined by i s p ima y s uc u e,
is e y o en he key ac o in delimi ing i s use in oods. I a p o ein has a pola
su ace due o he p esence o pola amino acids, i will ha e good solubili y
in a pola sol en such as wa e . On he o he hand, p o eins wi h highe
con en s o hyd ophobic amino acids, ewe cha ges on hei su ace, o hose
which con ain many subuni s end o ha e limi ed wa e solubili y
(Damoda an, Pa kin e al. 2007).
E ec o pH
Su aces o p o eins ha e ne cha ges due o hei amino acid con en
depending on he pH o hei en i onmen . A p o ein shows minimal solubili y
when i has a ne cha ge o ze o, ha is, equal numbe s o posi i e and
nega i e cha ges on i s su ace. This is called he isoelec ic poin o he
p o ein (IEP). The e is minimal solubili y because in e molecula epulsion is
a a minimum and p o eins will end o agg ega e. A pH alues abo e he
isoelec ic poin o a p o ein, i has a ne nega i e cha ge. A pH alues below
he isoelec ic poin , i will ha e a ne posi i e cha ge. In bo h cases he
p esence o p onounced su ace cha ges will esul in in e molecula epulsion
and enhanced solubili y.
Sal concen a ion’s
Solubili y is also a ec ed by he ype and concen a ion o sal s in a ood
sys em. As sal s con en inc eases p o eins become mo e soluble, which is he
so-called “sal ing-in” e ec , a ibu ed o he abili y o sal ions o enhance he
su ace cha ges on p o eins. In oods, sodium chlo ide is commonly used o
his pu pose. A high sal concen a ions, usually abo e 1M, which is much
highe han he concen a ion used in oods u s mola , p o ein solubili y
dec eases. This e ec is called “sal ing-ou ” and is hough o be due o sal
compe ing wi h he p o eins o a ailable wa e o sol a ion.
Backg ound. P o eins
53
Dena u a ion o p o ein
As desc ibed in sec ion 3.1.5, when dena u a ion p oceeds, p o ein
molecules change in ega d o su ace cha ges, shape, size, and
hyd ophobici y. Mos ho oughly dena u ed p o eins a e insoluble. In any
case, dena u a ion always esul s in loss o solubili y, and hen i is undesi able
in many ood sys ems whe e solubili y is impo an . Howe e , dena u a ion
may cause some desi able changes in some ood sys ems. The e o e, he
deg ee o p o ein dena u a ion equi ed depends on he ood applica ion, bu
ensu ing i s exhaus i e con ol o e ood p ocessing is essen ial.
Gela ion
A p o ein gel is a h ee-dimensional c oss-linked ne wo k o p o ein
molecules imbedded in an aqueous sol en . Mos gels a e e y high in wa e
con en (up o 95-98%), and s ill ha e cha ac e is ics o solid o igid ood
ma e ials (Damoda an, Pa kin e al. 2007).
Gela ion is based on he dena u a ion o p o eins, ollowed by hei
in e molecula associa ion o o m ma ices which ap wa e , a , and o he
ood ing edien s. The o ma ion o gels is in luenced by hea , pH, p essu e o
shea ing, and sol en condi ions. Food gels a e di ided in o wo ca ego ies:
he mally e e sible and he mally i e e sible gels (Damoda an 1997).
The mally i e e sible ( he mose ) gels
The mally i e e sible o he mose gels o m chemical bonds ha will
no b eak du ing ehea ing o he gel and hus emain igid i i is ehea ed.
Mos he mose gels a e he esul o p o ein un olding, ollowed by
agg ega ion o he molecules in o a c oss-linked ne wo k. Du ing his p ocess,
hea ed p o eins pa ially un old and o m agg ega es. As he “gel-poin ”
empe a u e is eached, hese agg ega es un old u he and apidly c oss-link
o o m a gel. This ne wo k is gene ally o med ia non-co alen bonds such

Backg ound. P o eins
54
as hyd ophobic in e ac ions and hyd ogen bonds. Occasionally, disulphide
bonds may be in ol ed.
High P essu e P ocessing (HHP) in ood p ocessing
The e ec o he high p essu e on ood p ese a ion was s udy ini ially by
Hopkins, Hi e and Wa son (1899). In 1899 some expe imen s we e ca ied ou
in Wes Uni e si y (Vi ginia), using high hyd os a ic p essu e o conse e juice,
mea and ui . These s udies demons a ed ha some mic oo ganisms could
be des oyed a e 10 min a 658 MPa. A ea ly 20 h cen u y, o he esea ch
p o ed ha egg whi e could be modi ied by high p essu e. Howe e , he
esea ch in his ield did no unde go any subs an ial p og ess un il signi ican
imp o emen s we e achie ed in he echnology o hyd aulic p esses. This ac
encou aged he enewed in e es o esea che s on HHP applica ions in he
eigh ies.
When a high p essu e is apply o a ood dipped in a liquid, he p essu e
placed on he sample is he same in all poin s. This is one o he ad an ages o
his p ocedu e, a oiding he di e ences ound in he he mal ea men . In
addi ion, compa ed o he he mal ea men s, p essu isa ion and
dep essu isa ion cycles a e as e , educing p ocessing ime.
As abo e men ioned, HHP can leads o he des uc ion o mic oo ganisms
wi hou ma kedly al e ing he as e and la ou o he nu ien con en o
oods. In gene al, bac e ia, which a e in he loga i hmic g ow h phase, a e he
mos sensi i e. Mode a e p essu es (300-600 MPa) in ol e he dea h o he
ege a i e cell. Usually a p essu e o 400 MPa is apply o 5 min o educe he
popula ion en imes (Hoo e , Me ick e al. 1989). To des oy bac e ia spo es,
a highe p essu e is needed, howe e i he p essu e is combined o a so
hea ing (60oC), spo es a e des oyed a abou 400 MPa. Thus, a combina ion
o high p essu e and so hea ing can suppose a syne gic e ec (Galazka and
Ledwa d 1995).
Backg ound. P o eins
55
The enzyme ac i i y s ongly depends on pH, composi ion, empe a u e
and p essu e. Some enzymes can be disabled a 100 MPa, howe e o he
needs highe p essu es (e en 1,000 MPa) (Cano, He nandez e al. 1997)
In addi ion o mic oo ganisms inac i a ion. HHP can also un old p o eins,
solidi y lipids, showing ad an ages in he p ese a ion o senso y (colou ,
as e, la ou , ex u e, e c.) and nu i ional p ope ies (Tewa i, Jayas e al.
1999). In addi ion, high p essu e can induce con o ma ional changes in
p o eins, which can in ol e impo an modi ica ions o hei echno- unc ional
p ope ies. As a consequence, p o eins can unde go agg ega ion and gela ion,
depending on he p essu e applied, as well as p o ein na u e, composi ion,
and en i onmen al ac o s such as pH o ionic o ce.
Because he agg ega ion and gela ion a e di ec ly ela ed o p o ein-
p o ein in e ac ions, HHP p ocessing will ha e a high in luence on p o ein
s uc u e, since applica ion o HHP may exe s a ma ked e ec on non-
co alen in e ac ions (elec os a ic, hyd ophobic and hyd ogen bonds). Thus,
HHP ea men may lead o a b eakdown o he e ia y and qua e na y
p o ein s uc u e o globula p o eins, bu i has a e y limi ed in luence on
seconda y s uc u e.
In gene al, low p essu es induce e e sible changes such as p o ein-
complex dissocia ion, ligands, and con o ma ional changes, whe eas high
p essu es (> 500 MPa) gene ally in ol e i e e sible p o ein modi ica ions
(He eman, Van Camp e al. 1997).
Wa e binding
Wa e -binding capaci y is he amoun o wa e ha is bound o e ained
by a p o ein unde well-de ined condi ions. Thus, wa e binding is an
impo an echno- unc ional p ope y o se e al easons:
Backg ound. P o eins
56
- Mos oods con ain high amoun s o wa e and i is necessa y o a oid
chemical changes ha migh cause he o ma ion o ee wa e o d ip
loss.
- Inc easing he amoun o wa e a p oduc can hold e ec i ely can
inc ease he p o i abili y o a gi en p oduc
- Bo h p oduc yield and senso y quali y a e highly dependen on he
p ope mois u e con en o a inished ood.
Wa e is usually bounded o he su ace o a p o ein by hyd ogen
bonding, which is some imes called dipole bonding. Hyd ogen bonding esul s
om wa e ’s in e ac ion wi h he R g oup o amino acids which a e dipoles.
Wa e bound o he su ace o p o eins in his manne is called “monolaye ”
wa e and is e y igh ly associa ed wi h he p o ein. O he wa e associa ed
wi h he p o ein o p o ein ma ices can be apped in capilla y s uc u es and
po es. I wa e is no associa ed wi h he monolaye on he p o ein su ace is
called ee wa e and mo es unhinde ed h oughou he ood sys em.
Wi h ega ds o ac o s which In luence wa e binding, he mos
impo an a e small pola molecules (e.g. suga s), empe a u e, sal con en
and pH. Small pola molecules and empe a u e will gene ally enhance wa e
binding by p o eins. In some cases empe a u e may induce o ma ion o
p o ein gels, which will enhance he binding o wa e by he sys em. Sodium
chlo ide binds o cha ged g oups on p o ein su aces and weakens
in e molecula bonds. This is a posi i e e ec in sys ems which u ilize muscle
ib es as pa o he s uc u al elemen s o he ood. Sal allows he muscle
p o eins o dis ance hemsel es om o he s wi hin he muscle ib e and hus
inc ease he numbe o si es o wa e o bind. The pH o a sys em ma kedly
in luences i s abili y o bind wa e . This is due o changes in he su ace
Backg ound. P o eins
57
cha ges on a p o ein as he pH is al e ed. Wa e binding is he lowes a he
isoelec ic poin (IEP) o a p o ein (Damoda an 1997).
Emulsi ica ion
An emulsion is a mix u e o wo immiscible liquids in which one is
dispe sed in he o he in he o m o d ople s. The liquid in he d ople s he
dispe sed is called in e nal, o discon inuous phase. In he same way, he
su ounding phase is called he ex e nal o con inuous phase. Emulsions in
which he dispe sed phase is a lipid a e called “oil in wa e ” emulsions (O/W).
By he con a y, wa e in oil emulsions con ain d ople s o wa e dispe sed in
a lipid as con inuous phase (W/O) (McClemen s 2004).
When a liquid is exposed o o he phase (e.g. ai ), he su ace be ween
hem is in a s a e o ension. This s a e o ension is called in e acial ension
(o su ace ension, in he case o ai ) and i is a consequence o he a ac i e
o ces be ween molecules in he liquid ha a e enhanced by exposu e o he
o he phase. The molecules “bunch” oge he o dec ease hei exposi ion o
he ai su ace. The egion o con ac be ween wo immiscible liquids is called
he in e ace. The in e acial a ea plays an impo an ole in emulsion
o ma ion. Thus, a conside able amoun o mechanical ene gy is equi ed in
o de o educe d ople size and o inc ease in e acial a ea (Wals a 1993).
Howe e , as he in e acial a ea inc eases, he s abili y o he mix u e
dec eases. Un o una ely, mos emulsions a e he modynamically uns able
and d ople s end o agg ega e spon aneously in o de o educe he
in e acial a ea. The e o e, emulsion s abili y, which is conside ed o be he
p ima y equi emen o he comme cial applica ion o emulsions, is in ac a
kine ic concep such ha an emulsion is conside ed s able when he numbe ,
size dis ibu ion, and a angemen o d ople s do no unde go any disce nible
change o e he s o age ime scale.
Backg ound. P o eins
64
Wa e Re en ion
The abili y o mea and mea p oduc s o e ain mois u e be o e, du ing,
and a e p ocessing o cooking plays a c ucial ole in consume accep ance o
he p oduc and is usually desc ibed in e ms o wa e -holding capaci y.
Physico-chemically, he wa e in mea is p esen in ei he he bound o he
ee s a e. The bound wa e is igh ly associa ed wi h p o eins h ough
cha ged g oups and dipola si es on he p o ein su ace.
Solubili y
Solubili y o p o eins is qui e impo an o he manu ac u e o p ocessed
muscle oods. This is because mos echno- unc ional p ope ies o muscle
p o eins a e ela ed o p o ein solubili y, and, in ac , some a e achie ed only
when he p o eins a e in a highly soluble s a e. This is because mos unc ional
p ope ies o muscle p o eins a e ela ed o p o ein solubili y, and, in ac ,
some a e achie ed only when he p o eins a e in a highly soluble s a e.
Solubili y o muscle p o eins is a unc ion o p o ein s uc u es, he
s uc u e o myo ib ils, pH, concen a ion (ionic s eng h) o sal added o
mea , empe a u e, ime o mixing mea wi h sal ... Sa coplasmic p o eins a e
na u ally soluble in muscle. Howe e , solubilisa ion o myo ib illa p o eins
gene ally equi es ela i ely high ionic s eng h (G > 0.4 M). Thus, p o ein
solubili y is highly dependen on he ionic s eng h o he ex ac ion bu e .
Ex ac ion o myo ib illa p o eins begin a an ionic s eng h close o 0.5 M,
and i eaches a maximum a ionic s eng h 1.0 M. Thus, an inc ease in sal
(NaCl) concen a ion o abo e 0.5 M (app oxima ely 2% sal in mea ), is widely
used in p ocessed mea s (Xiong 2004).
Viscosi y o p o ein solu ions
Rheological p ope ies, as ela ed o low and de o ma ion, a e impo an
unc ional a ibu es o muscle p o eins. The heological beha iou o a

Backg ound. P o eins
65
p o ein suspension in muscle oods is o en desc ibed in e ms o iscosi y.
This is because he heological p ope ies o he aqueous p o ein phase can
in luence ex u e and s abili y.
P o eins a e cha ged polyme s capable o binding wa e and causing ib e
swelling by he up ake o wa e and loosening o he polypep ide ma ix. As a
consequence o swelling p ocess, a p o ein inc eases i s e ec i e
hyd odynamic olume, and, he e o e, inc eases he esis ance o shea .
Finally, he myosin s uc u e ( he la ge leng h- o-diame e a io o he
od po ion), makes myosin highly iscous in sal solu ion. Because o i s g ea
iscosi y and abundance in muscle, myosin is he majo con ibu o o he
heological p ope ies o he aqueous ex ac in sal ed mea (Xiong 2004).
Gela ion
Gela ion o p o eins is a he modynamic p ocess ha occu s widely in
ood p ocessing using muscle p o eins. A gel has been e e ed o as a
con inuous ne wo k o mac oscopic dimensions imme sed in a liquid medium
and exhibi ing non s eady s a e low. The impo ance o p o ein gela ion o
muscle oods has been demons a ed. Thus, myo ib illa p o eins a he
junc ion o mea pa icles we e esponsible o he mea binding and ex u e
o cooked sausage p oduc s (Xiong 2004).
Emulsi ica ion
Emulsions om muscle p o eins a e s abilized h ough wo mechanisms.
The i s mechanism is physical en apmen o a globules wi hin he p o ein
ma ix o med la gely ia p o ein-p o ein in e ac ions. In he second
mechanism, a globules a e s abilized by an in e acial p o ein ilm
(memb ane) ha su ounds hem. The in e acial ilm is in e ac i e in he
sense ha i in e ac s wi h he iscoelas ic con inuous phase o u he
enhance he emulsion s abili y.
Backg ound. P o eins
66
In mea emulsions, sal -soluble p o eins play he mos c i ical ole in
o ming in e acial ilms ha encapsula e a pa icles o oil d ople s. The
emulsi ying capaci y o di e en muscle p o eins was ound o ollow he
o de o myosin > ac omyosin > sa coplasmic p o eins > ac in (Xiong 2004).
Foaming
Foaming o p o ein solu ions is ai ly common. Di icul ies a ise when he
oam olume expands o he capaci y o he con aine and p o eins become
dena u ed as a esul o oam o ma ion. The beha iou o p o eins a he
ai /liquid in e ace is ex emely impo an because he o ma ion o a p o ein-
based lexible, cohesi e ilm a ound ai bubbles is essen ial o oaming
capaci y and oam s abili y. In ac , he e is a ela ionship be ween he
molecula lexibili y o p o eins, ilm p ope ies, and oam s abili y. Flexible,
diso de ed p o eins a e mo e su ace-ac i e han ex ensi ely c oss-linked,
s able, and compac globula p o eins (Xiong 2004).
Backg ound. In e acial Assessmen
67
3.2. Rheology
De ini ion
The e m " heology" is o igina ed om he G eek: " heos" meaning " he
i e ", " lowing", "s eaming". Thus, heology is li e ally " he science o low".
Mo e speci ically, heology is he science o low and de o ma ion o ma e
and desc ibes he in e ela ion be ween o ce, de o ma ion and ime.
The e o e, i is a b anch o physics since hese a iables come om he ield
o mechanics. Howe e , heological expe imen s do no me ely show
in o ma ion abou he low beha iou o liquids, bu also abou he
de o ma ion beha iou o solids (Mezge 2014).
Flow laws
Isaac New on was he i s o exp ess he basic law o iscosi y desc ibing
he low beha iou o an ideal liquid. Thus, New on pos ula ed ha an ideal
(New onian) luid is a luid in which he iscous s esses a ising om i s low,
a e e y poin , a e linea ly p opo ional o he local s ain a e. The cons an
o p opo ionali y in New on's Law is he iscosi y o he luid. EQ. (3.2-1)
illus a es his beha iou :
𝜏=𝜂∙𝛾󰇗
(3.2-1)
whe e τ ep esen s he s ess, η he iscosi y and 𝛾󰇗 he s ain a e
Usually, a simple shea expe imen , in which a luid is con ined be ween
wo pla es (Figu e 3.2-1) is used o de ine some undamen al heological
pa ame e s (Two Pla es Model). The uppe pla e wi h he (shea ) a ea A is se
in mo ion by he (shea ) o ce F and he esul ing eloci y max is measu ed.
The lowe pla e is s a iona y ( = 0). The gap size (y1 o y2) is he dis ance
be ween he pla es, and he liquid sample is shea ed wi hin his gap. I is
assumed ha he ollowing shea condi ions a e me :
Backg ound. In e acial Assessmen
68
1) The sample adhe es o bo h pla es and does no slide o slip along
hem.
2) The e a e lamina low condi ions.
Figu e 3.2-1: Two-pa e model ep esen a ion
Shea s ess
A o ce applied angen ially o an a ea being he in e ace be ween he
uppe pla e and he liquid unde nea h, leads o a low in he liquid laye . Shea
s ess (τ) p ecisely a ises om he applica ion o his angen ial o ce (F) o he
shea su ace a ea (A), as he a io be ween bo h a iables:
𝜏=𝐹
𝐴
(3.2-2)
Shea a e
The shea s ess (τ) causes he liquid o low in a special pa e n. A
maximum low speed is ound a he uppe bounda y. The speed d ops ac oss
he gap size (y) down o 0 ( min = 0) a he lowe bounda y con ac ing he
s a iona y pla e. Lamina low means ha in ini esimally hin liquid laye s slide
on op o each o he , simila o he ca ds in a deck o ca ds. Thus, one lamina
laye is hen displaced wi h espec o he adjacen ones by a ac ion o he
o al displacemen encoun e ed in he liquid be ween bo h pla es. The speed
Backg ound. In e acial Assessmen
69
d op ac oss he gap size is named “shea a e” (γ ) and i may be
ma hema ically de ined by a di e en ial unc ion o he luid eloci y ( ), as
ollows:
𝛾󰇗=𝑑𝑣
𝑑𝑦
(3.2-3)
In addi ion, he shea a e may be de ined as he ime-de i a i e o he
s ain caused by he shea s ess ac ing on he liquid laye :
𝛾󰇗=𝑑𝛾
𝑑𝑡
(3.2-4)
The e o e, EQ. (3.2-3) and EQ. (3.2-5) may be combined o gi e a mo e
gene al exp ession o New on:
𝜏=𝜂∙𝑑𝑣
𝑑𝑦= 𝜂∙ 𝛾󰇗
(3.2-5)
Dynamic iscosi y
F om EQ. (3.2-1), he dynamic iscosi y can be eadily w i en as ollows:
𝜂=𝜏𝛾󰇗
(3.2-6)
Fo ideal- iscous luids measu ed a a cons an empe a u e, he alue o
he a io o he shea s ess (τ) o he co esponding shea a e 𝛾󰇗 is a ma e ial
cons an (η) (New onian luids).
Non-New onian liquids. Non-ideal liquids
The e a e se e al kind o luids which do no exhibi he ideal New onian
beha iou whe e η is no cons an . Va ious ypes o common low beha iou
can be obse ed in Figu e 3.2-2.

Backg ound. In e acial Assessmen
70
Figu e 3.2-2: Beha iou o di e en luids: New onian (1), Pseudoplas ic o Shea -
hinning (2), Dila an o Shea hickening (3) and Pseudoplas ic o Shea - hinning
luid wi h a yield poin (4).
Pseudoplas ic luids
Many liquids show d as ic iscosi y dec eases when he shea a e is
inc eased om low o high le els. This means ha o a gi en o ce mo e mass
can be made o low o he ene gy can be educed o sus ain a gi en low a e
(phenomena desi able in indus ial p ocesses).
This beha iou is ela ed o hei in e nal s uc u e. Many liquid p oduc s
ha seem homogeneous a e in ac composed o se e al ing edien s ha may
possess i egula shapes o may in e ac wi h each o he . O he liquids may
consis o polyme solu ions wi h long en angled and looping molecula
chains. A es , all o hese ma e ials will main ain an i egula in e nal o de
and co espondingly hey a e cha ac e ized by a sizable in e nal esis ance
agains low (and as a consequence high iscosi y). Wi h inc easing shea
a es, ma chs ick-like pa icles suspended in he liquid will be u ned
leng hwise in he di ec ion o he low. Chain- ype molecules in a mel o in a
solu ion can disen angle, s e ch and o ien hemsel es pa allel o he d i ing
o ce. Pa icle o molecula alignmen s allow pa icles and molecules o slip
pas each o he mo e easily. Sphe ical pa icles may be de o med o o al-
shaped pa icles (smalle in diame e bu longe ). Mo eo e , he e a e se e al
Backg ound. In e acial Assessmen
71
o he possible explana ions o his beha iou , e. g. sol en laye s may be
s ipped om dissol ed molecules o om pa icles, which means, ha he
in e molecula in e ac ions causing esis ance o low become educed.
Finally, o mos liquid ma e ials he shea - hinning e ec is e e sible
(Mezge 2014) .
Dila an luids
Fluids a e liquids which unde ce ain condi ions o s ess o shea a e
inc ease hei iscosi y whene e shea a e inc eases. Thus, he esis ance o
low inc ease and may become so high ha make impossible o pomp ou he
luid.
Dila an low beha iou is ela i ely common in highly concen a ed
suspensions. The pa icles a e densely packed and he amoun o liquid is jus
su icien o ill he space be ween he pa icles. A es o a low low a e he
liquid medium ully lub ica es he pa icle su aces and hus allows an easy
posi ional change o pa icles when o ces a e applied ( his suspension
beha es as a liquid a low shea a es). A highe shea a es, pa icles will
wedge o he s, causing gene al olume inc eases. Since he liquid is no longe
su icien o ill all oids and o keep he pa icle su aces ully lub ica ed, he
solu ion becomes mo e iscous. Howe e , dila an luids a e a e and his low
beha iou mos likely complica es p oduc ion condi ions (Sch amm 2000).
Yield poin
A sample wi h a yield poin begins o low only i he ex e nal o ces Fex
ac ing on he ma e ial a e la ge han he in e nal s uc u al o ces Fin . Below
he yield poin he ma e ial shows elas ic beha iou , exhibi ing unde load
only a e y small deg ee o de o ma ion ha does no emain a e emo ing
he load. Thus, When Fex < Fin he ma e ial is only de o med o a small deg ee.
The sample does no begin o low be o e Fex > Fin . Finally, he yield poin is
Backg ound. In e acial Assessmen
72
also e e ed o as yield s ess o yield alue (Sch amm 2000). The e is a wide
ange o ma e ials which appea o show his kind o beha iou , howe e
Ba nes (2000) s a ed ha he e is as much happening in e ms o low below
as abo e he ‘yield s ess’. Thus, Ba nes showed a numbe o examples o such
liquids, whe e he iscosi y alls many o de s o magni ude o e a na ow
ange o shea s ess, and indeed when app oaching his c i ical s ess egion
om egions o high s ess i appea s ha he iscosi y goes o in ini y.
Howe e , ca e ul and pa ien measu emen below his s ess shows ha he
iscosi y is s ill ini e, and e en ually le els o o a cons an , bu e y high
alue a low s esses. Fluids exhibi ing such beha iou we e named “ e y
shea - hinning” o “yield s ess” luids and he c i ical s ess was deno ed as
“appa en yield s ess” (Ba nes 2000).
Thixo opic luids
Thixo opy is he change o iscosi y wi h ime o shea ing, and is
gene ally iewed as a oublesome p ope y ha one could well do wi hou .
In ac , hixo opy could be be e seen as he esul o a high deg ee o shea
hinning, and comes abou whene e a shea -induced change in
mic os uc u e akes ime o occu .
Mic os uc u e is b ough o a new equilib ium by compe i ion be ween,
on he one hand he p ocesses o ea ing apa by s ess du ing shea ing, and
on he o he hand build-up due o low and B ownian mo ion induced
collision, o e a ime ha can be minu es. Then, when he low ceases, he
B ownian mo ion ( he only o ce le ) is able o slowly mo e he elemen s o
he mic os uc u e a ound o mo e a ou able posi ions and hus ebuild he
s uc u e: his can ake many hou s o comple e. The whole p ocess is
comple ely e e sible.
Backg ound. In e acial Assessmen
73
Thixo opy is a unc ion o ime and shea a e, and he e o e canno be
p ope ly accoun ed o in expe imen s whe e bo h hese a iables a e
changed simul aneously. As a consequence, he bes expe imen s o p ope ly
measu e hixo opy a e hose whe e he sample o be es ed is shea ed a a
gi en shea a e un il equilib ium is ob ained (Ba nes 2000).
Elas ic Beha iou
Nowadays ins umen s allow o cha ac e ize elas ic beha iou in a ange
o e y low de o ma ions, and he e o e wi hou any des uc ion o he
s uc u e o ma e ials o be es ed.
In o de o de ine u he heological pa ame e s, Two Pla es Model will
be used. This model is plo ed in Figu e 3.2-3, whe e shea s ain is de ined as
ollows:
Figu e 3.2-3: Shea de o ma ion o a ma e ial using he Two Pla es Model.
Shea modulus
When measu ing an ideal-elas ic solid a a cons an empe a u e, he
a io o he shea s ess and he co esponding de o ma ion is cons an i he
measu emen akes place wi hin he e e sible elas ic de o ma ion ange
F
h
s
A
ϕ
𝛾=𝑠ℎ
(3.2-7)
Backg ound. In e acial Assessmen
80
Ini ially, he e is an ini ial elas ic esponse. The ea e he e is a so-called
delayed elas ic esponse whe e he de o ma ion a e becomes slowe and
slowe , ending up as a e y slow bu s eady-s a e de o ma ion a he longes
imes. This cu e desc ibes he c eep esponse o mos s uc u ed liquids and
gels.
Models o c eep es
In gene al, in a c eep es , a simple elas ic solid (a sp ing) shows an
immedia e esponse o gi e a cons an de o ma ion (s ain). On he o he
hand, a simple New onian liquid (a dashpo ) would show an e e -inc easing
s ain, which displayed on a g aph o s ain agains ime would be a s aigh
line s a ing a he o igin, wi h he slope gi ing he shea a e 𝛾󰇗.
Maxwell Model
Thus, acco ding o Maxwell model, he beha iou can be desc ibed by he
EQ. (3.2-15).
𝛾=𝜏(1
𝐺+𝑡𝜂)
(3.2-15)
whe e, as de ined p e iously, he s ain is gi en by γ, he modulus by G,
he ime by and he iscosi y by η.
Thus, a e y sho imes, is cha ac e ised by an immedia e elas ic
esponse, (γ = τ/G) and a e y long imes, when >> η/G, by simple iscous
beha iou , γ = σ /η. He e η/G is called he elaxa ion ime λ.
Kel in-Voig model
I a c eep es is pe o med on a Kel in-Voig model, he s ain g adually
builds up o a cons an alue as desc ibed by EQ. (3.2-16), which is he solu ion
o he model o he c eep es .

Backg ound. In e acial Assessmen
81
𝛾=𝜏
𝐺[1−𝑒−𝑡 𝜆′
⁄]
(3.2-16)
He e, λ’ is called he e a da ion ime, since i cha ac e ises he e a ded
esponse o he model, and i s alue is again gi en by η/G. A e y sho imes,
he esponse is iscous, and 𝛾~𝑡∙ 𝜏/𝜂.
Models o elaxa ion es s
Relaxa ion es s consis on he applica ion o a cons an s ain, and he
moni o ing o he consequen s ess, which hen decays away wi h ime.
Maxwell Model
Thus, acco ding o Maxwell model, he s ain and he shea a e is as
ollows:
𝛾=𝛾𝑒𝑙+𝛾𝑣𝑖𝑠
(3.2-17)
𝛾󰇗=𝛾󰇗𝑒𝑙+𝛾󰇗𝑣𝑖𝑠
(3.2-18)
A e mul iplying bo h equa ion sides by μ, we can ob ain:
𝜇𝛾󰇗 =𝜇𝛾󰇗𝑒𝑙+𝜏
(3.2-19)
Taking in o accoun ha 𝛾 󰇗𝑒𝑙=𝐺−1·𝑑𝜏 𝑑𝑡
⁄, EQ. (3.2-19) can be exp essed as
ollows:
𝜏=𝛾󰇗−𝜆𝑑𝜏
𝑑𝑡
(3.2-20)
Kel in-Voig Model
Thus, acco ding o Kel in-Voig model, he s ess applied is as ollows:
𝜏=𝜏𝑒𝑙+𝜏𝑣𝑖𝑠
(3.2-21)
Backg ound. In e acial Assessmen
82
In oducing EQ. (3.2-11) and EQ. (3.2-12) in EQ (3.2-21), we can ob ain he
ollowing exp ession:
𝜏=𝐺·𝛾+𝜇𝛾󰇗
(3.2-22)
A e di iding bo h equa ion sides by G, we can ob ain:
𝜏
𝐺=𝛾+𝜆′𝑑𝛾
𝑑𝑡
(3.2-23)
Oscilla o y es s
Oscilla o y es s a e used o examine all kinds o iscoelas ic ma e ials,
om low- iscosi y liquids o polyme solu ions, and e en igid solids. This
mode o es ing is also e e ed o as "dynamic mechanical analysis" (DMA).
To explain oscilla o y es s, he Two Pla es Model is used again. The
bo om pla e is s a iona y. When he wheel o disk is u ning, he uppe pla e
wi h he (shea ) a ea is mo ed back and o h by he (shea ) o ce. Figu e 3.2-9
ep esen s his mo emen :
Figu e 3.2-9: Oscilla o y es s o a luid be ween wo pa allel pla es
whe e F is he shea o ce, S he de lec ion pa h, ϕ he de lec ion angle
and h he shea gap.
Thus, ins ead o applying a cons an s ess leading o a s eady-s a e low,
samples a e subjec ed o oscilla ing s esses o oscilla ing s ains. The s ess
may be applied as a sinusoidal ime unc ion (EQ. 3.2-24):
s s
h
-F +F
ᵩ
ᵩ
Backg ound. In e acial Assessmen
83
𝜏=𝜏0∙sin (𝜔𝑡)
(3.2-24)
whe e τ is he s ess applied, which is a sinusoidal unc ion o he
maximum s ess applied (τ0), as well as, he equency (ω) and ime ( ).
Theo e ical aspec s o dynamic es ing
The Hookean Sp ing in oscilla o y mo emen
Figu e 3.2-10 ep esen s he mo emen o a wheel connec ed o he
sp ing model.
Figu e 3.2-10: Sp ing subjec ed o an oscilla o y mo emen
The sp ing ex ends o a maximum s ain (γ0) and con ac s o i s o iginal
leng h wi h a equency equal o he angula eloci y o he wheel (ω) and
hen he s ain and he s ess can be w i en as a unc ion o ime as ollows:
𝛾=𝛾0∙sin (𝜔𝑡)
(3.2-25)
Thus, he s ess unc ion is de ined by EQ. (3.2-26):
𝜏=𝐺𝛾0∙sin (𝜔𝑡)
(3.2-26)
Fo his case s ain and s ess a e in-phase wi h each o he . Tha is, upon
de o ma ion, he maximum s ess and he maximum s ain occu a he same
ins an in ime.
The new onian dashpo model in oscilla o y mo emen
Figu e 3.2-11 ep esen s he mo emen o a wheel connec ed o he
dashpo model:
Backg ound. In e acial Assessmen
84
Figu e 3.2-11: Dashpo model in an oscilla o y mo emen
Consequen ly, i he sp ing is exchanged by a dashpo and he pis on is
subjec ed o a simila c anksha ac ion, he ollowing equa ions apply:
𝛾󰇗=𝑑𝛾
𝑑𝑡=ω ∙ 𝛾󰇗∙ cos (𝜔𝑡)
(3.2-27)
Subs i u ing his in o he dashpo equa ion:
𝜏=𝜂∙𝑑𝛾
𝑑𝑡=𝜂∙𝜔∙𝛾0∙cos(𝜔𝑡)
(3.2-28)
Fo he dashpo he esponse o τ is 90° ou o phase o he s ain. This
can also be exp essed by de ining a phase shi angle δ = 90° by which he
assigned s ain is ailing he measu ed s ess.
The abo e equa ion EQ. (3.2-28) can hen be ew i en:
𝜏=𝜂∙𝜔∙𝛾0∙cos(𝜔𝑡)=𝜂∙𝜔∙𝛾0∙sin(𝜔𝑡+𝛿)
(3.2-29)
Acco ding o his equa ion, in-phase s ess esponse o an applied s ain
is called “elas ic”. A 90° ou -o -phase s ess esponse is called “ iscous”.
I a phase shi angle is wi hin he limi s o 0 < d < 90° is called “ isco-
elas ic”.
The Maxwell model in oscilla o y mo emen
Figu e 3.2-12 ep esen s he mo emen o a wheel connec ed o he
Maxwell model:
Backg ound. In e acial Assessmen
85
Figu e 3.2-12: Maxwell model in an oscilla o y mo emen
The s esses in each elemen a e equal and he o al s ain is he sum o
he s ains in bo h he dashpo and he sp ing.
The equa ion o s a e o he model is EQ. (3.2-30):
1
𝐺∙(𝑑𝜏
𝑑𝑡)+𝜏𝜂=𝑑𝛾
𝑑𝑡
(3.2-30)
In oducing he sinusoidal unc ion, we can ob ain EQ. (3.2-31).
1
𝐺∙(𝑑𝜏
𝑑𝑡)+𝜏𝜂=𝜔∙𝛾0∙cos(𝜔𝑡)
(3.2-31)
The s ess esponse o he sinusoidal s ain consis s o wo pa s which
con ibu e he elas ic sin-wa e unc ion wi h ϕ = 0° and he iscous cosine-
wa e- unc ion wi h ϕ = 90°
The Kel in-Voig model in oscilla o y mo emen
Figu e 3.2-13 ep esen s he mo emen o a wheel connec ed o he
Kel in-Voig model:
Figu e 3.2-13: Kel in-Voig model in an oscilla o y mo emen

Backg ound. In e acial Assessmen
86
This model combines a dashpo and sp ing in pa allel. The o al s ess is
he sum o he s esses o bo h elemen s, while he s ains a e equal.
The equa ion o s a e o he model is EQ. (3.2-32).
𝜏=𝐺∙𝛾+𝜂∙𝑑𝛾
𝑑𝑡
(3.2-32)
In oducing he sinusoidal unc ion, we can ob ain EQ. (3.2-33).
𝜏=𝐺∙𝛾0∙sin(𝜔𝑡)+𝜂∙𝜔∙𝛾0∙cos(𝜔𝑡)
(3.2-33)
The s ess esponse in his wo-elemen -model is gi en by wo elemen s
being elas ic when δ = 0, and being iscous when δ = 90°.
Real iscoelas ic samples
Real iscoelas ic samples a e mo e complex han ei he he Kel in-Voig
solid o he Maxwell liquid. Thei phase shi angle is posi ioned be ween 0 <
δ < 90°. G and δ a e again equency dependen :
I is common o in oduce he e m Complex Modulus (G*) which is
de ined as EQ. (3.2-34) indica es:
𝐺∗=𝜏0
𝛾0
(3.2-34)
G* ep esen s he o al esis ance o a subs ance agains he applied
s ain. I is qui e impo an o no e ha o eal iscoelas ic ma e ials bo h he
complex modulus (G*) and he phase angle (γ) a e equency dependen .
Complex numbe s can be used o exp ess he complex modulus in wo
o he pa ame e s. Thus, Complex modulus (G*) can be de ined as EQ (3.2-35)
shows:
𝐺∗=𝐺′+𝑖𝐺′′=𝜏0(𝑡)
𝛾𝐸 (𝑡)
(3.2-35)
Backg ound. In e acial Assessmen
87
whe e he eal pa o he complex unc ion is he elas ic componen , G’,
and he imagina y pa co esponds o he iscous componen , G’’. As a
consequence, he complex modulus G* (|G*|) esul s om he combina ion
o bo h pa s:
|𝐺∗|=√𝐺′2+𝐺′′2
(3.2-36)
Thus, G’ and G’’ may be exp essed in e ms o he measu ed quan i ies
(τ0 and γ0) and he phase angle be ween hem (δ):
𝐺′=𝐺∗∙cos𝛿=𝜏0
𝛾0∙ cos𝛿
(3.2-37)
𝐺′′=𝐺∗sin𝛿=𝜏0
𝛾0∙sin𝛿
(3.2-38)
I a subs ance is pu ely iscous hen he phase shi angle d is 90°: G’ = 0
and G’’ = G*. On he con a y, i he subs ance is pu ely elas ic hen he phase
shi angle d is ze o: G’ = G* and G’’ = 0 (Sch amm 2000).
I is also use ul o de ine he an δ EQ. (3.2-39).
an𝛿=𝐺′′
𝐺′
(3.2-39)
G' is known as he s o age modulus. G’ is a measu e o he de o ma ion
ene gy s o ed by he sample du ing he shea p ocess. A e he load is
emo ed, his ene gy is comple ely a ailable, now ac ing as he d i ing o ce
o he e o ma ion p ocess which pa ially o comple ely compensa es he
p e iously ob ained de o ma ion o he s uc u e. Ma e ials which a e s o ing
o be whole de o ma ion ene gy a e showing comple ely e e sible
de o ma ion beha iou since hey a e occu ing inally wi h an unchanged
shape a e a load cycle. Thus, G' ep esen s he elas ic beha iou o a
ma e ial.
Backg ound. In e acial Assessmen
88
On he o he hand, G" is known as he loss modulus. G" is a measu e o
he s ess ene gy used by he sample du ing he shea p ocess and he e o e,
i is e en ually los by ans o ma ion in o hea . Ene gy losing ma e ials show
i e e sible de o ma ion beha iou since hey occu wi h a change o shape
a e a load cycle. Thus, G" ep esen s he iscous beha iou o a ma e ial
(Mezge 2006).
Al e na i ely o he complex modulus G* one can de ine a complex
iscosi y, as EQ. (3.2-40) indica es:
𝜂∗=𝐺∗
𝑖𝜔=𝜏0
𝛾0𝜔
(3.2-40)
The complex iscosi y (η^*) desc ibes he o al esis ance o a dynamic
shea . I can again be b oken in o he wo componen s o he s o age iscosi y
η’ ( he elas ic componen ) and he dynamic iscosi y η’’ ( he iscous
componen .
The oscilla o y esponse o eal sys ems
The mos gene al esponse o G’ and G’’ o eal samples ( o s uc u ed
sys ems) is shown in Figu e 3.1-1. The exac alues o he moduli and hei
posi ion in he equency domain will a y, howe e , his igu e indica es he
o e all quali a i e beha iou . A numbe o speci ic egions can o en be
di e en ia ed, namely:
a) The iscous o e minal egion, whe e G’ p edomina es and iscous
( low) beha iou p e ails. All ma e ials ha e such a egion, e en solids
(because hey c eep a long imes), bu he equency whe e his is
seen is o en so low ha mos oscilla o y ins umen s canno de ec
his pa o he cu e.
Backg ound. In e acial Assessmen
89
b) The ansi ion- o- low egion is so called because, when iewed om
highe equencies (whe e elas ic beha iou domina es and G’ > G’’),
he loss modulus G’’, desc ibing iscous o low beha iou , becomes
signi ican . The poin whe e he wo moduli c oss o e is some imes
no ed, and o a Maxwell model, his c osso e equency is gi en by
he in e se o he elaxa ion ime.
c) The ubbe y o pla eau egion is whe e elas ic beha iou domina es.
While in many cases we see wha appea s o be a la pla eau, he e
is always a sligh inc ease o G’ wi h equency, bu i can be as small
as a ew pe cen inc ease in modulus pe decade inc ease in
equency. The alue o G’’ is o cou se always lowe han ha o G’,
bu some imes i can be conside ably lowe .
Figu e 3.2-14: Regions in he iscoelas ic spec um o non-New onian liquids.
d) A lea he y o highe ansi ion c osso e egion is also seen, whe e,
due o high- equency elaxa ion and dissipa ion mechanisms, he
alue o G’’ again ises, his ime as e han G’. Once mo e a G’ = G’’,
a c osso e equency can be de ined, om which ano he
cha ac e is ic ime can be ob ained.
Backg ound. P oduc s om p o eins
96
amoun , he mo e space ha he p o ein molecule has o sp ead ou a he
su ace, and hence he g ea e he oppo uni y o un olding o minimize he
con igu a ional ee ene gy ollowing adso p ion. Ea ly adso bing p o eins
end o exhibi a la ge loss o enzyma ic ac i i y, and a e poo ly exchangeable
wi h he bulk phase a e adso p ion. La e adso bing p o eins end o e ain
mo e enzyma ic ac i i y due o less un olding and mo e pa icipa ion in loosely
held mul ilaye s.
The maximum adso bed amoun is de e mined by he a e o un olding
a he su ace in ela ion o he a e o adso p ion. Fas adso p ion gi es less
ime o p o ein molecules o sp ead ou a he su ace, and consequen ly he
a ea occupied pe molecule is lowe and he adso bed amoun is highe .
Adso bed laye s a luid in e aces
Almos e e y hing desc ibed p e iously o p o ein adop ion a solid
su ace is applicable when p o eins a e adso bed a luid- luid in e aces.
Howe e , he e a e some di e ences. In pa icula , ela ed o how p o ein
molecules can pene a e u he in o he non-aqueous phase and can mo e
mo e eely, on a liquid su ace.
P ocesses in ol ing di usion, eo ien a ion and con o ma ional
eo ganiza ion will occu as e a ai -wa e and oil-wa e in e aces han on
solid subs a es. In addi ion, ano he impo an di e ence is ha in luid- luid
in e aces is possible o pe o m addi ional expe imen al s udies on he
su ace equa ion o s a e and on he su ace heological beha iou .
Analy ical su ace equa ions o s a e de eloped o small-molecule
adso bed laye s a e unable o ep esen ully he p ope ies o p o ein
sys ems. Howe e , some simple o mula can cap u e mos o he essen ial
ea u es o he beha iou . Nea ly all p o ein adso p ion s udies a e
cha ac e ized by ex emely non-ideal beha iou . The non-ideali y a ises om

Backg ound. P oduc s om p o eins
97
a combina ion o en halpic and en opic con ibu ions o he su ace ee
ene gy as a esul o complex in e molecula in e ac ions and in amolecula
ea angemen s. F umkin (1925) desc ibed a ai ly simply equa ion equa ion
ha can accoun o bo h en halpic and en opic e ms:
Π𝜔1
𝑅𝑇−ln(1−𝜃)−(1−𝑆−1)𝜃−𝐻
𝑅𝑇𝜃2
(3.3-3)
whe e S is he a io (ω1/ω2) o he sol en mola a ea (ω1) o he p o ein
mola a ea (ω2), H is he en halpy o mixing o a egula solu ion, R is he gas
cons an , T is he empe a u e and θ is he ac ional su ace co e age which
may be exp essed as 𝜃=𝜔2𝛤, in e ms o he su ace load (Γ).
The i s e m is he su ace p essu e o an ideal su ace mix u e o equal-
sized adso bed molecules. The second e m (linea in θ) allows o he non-
ideal su ace en opy o mixing o la ge and small adso bed molecules ( o
S<<1). This e m has he e ec o g ea ly educing he alue o Π, especially a
low alues o θ. The hi d e m (p opo ional o θ2) is ela ed o in e molecula
in e ac ions in he adso bed laye . Fo he no mal case o ne a ac i e
p o ein-p o ein in e ac ions (H>0), he combina ion o subs an ial en halpy
and en opy con ibu ions ep oduces he e y s ong de ia ion om ideali y
ypically obse ed a low su ace co e age.
Howe e , his equa ion b eaks down a high su ace co e age (θ→1)
when θ is p edic ed o s ongly di e ge, whe eas in p ac ice Π eaches a
sa u a ion alue co esponding o monolaye collapse and possible onse o
mul ilaye o ma ion. The heo y b eaks down because i is based on a wo-
dimensional model o he adso bed laye , whe eas he eal sys em is h ee-
dimensional.
Rega dless o he es ic ion o he wo-dimensional model, o he
e inemen s can in p inciple be made o accoun o p o ein agg ega ion and
Backg ound. P oduc s om p o eins
98
eo ien a ion in he adso bed laye . In pa icula , a mo e ealis ic allowance
o un olding o p o ein molecules in he adso bed laye can be made by
eplacing he single s a e o he adso bed p o ein by a dis ibu ion o s a es
wi h di e en alues o he mola a ea ω2. Howe e , such complex models
ha e he disad an age o inc easing he numbe o adjus able pa ame e s
wi hou p o iding a ully igo ous s a is ical mechanical desc ip ion o he
sys em unde conside a ion (Dickinson and McClemen s 1995).
Simula ion model o a globula p o ein adso bed
laye
A compu e simula ion model is a ailable in which an adso bed
monolaye o globula p o ein molecules a a luid– luid in e ace is
ep esen ed as a quasi- wo-dimensional ne wo k o c oss-linked igid
sphe ical pa icles (Wijmans and Dickinson 1998).
The app oach is mo i a ed by he simila i y in heological p ope ies
be ween a globula p o ein adso bed ilm and an ex emely hin laye o bulk
hea -se p o ein gel. The monolaye ne wo k s uc u es p oduced by he
model a e quali a i ely simila o hose gene a ed in wo-dimensional
simula ions o agg ega ed pa icle gels o med om i e e sibly bonding
B ownian pa icles. Howe e , he B ownian dynamics algo i hm used o
simula e he adso bed laye p ope ies is ac ually close in de ail o ha used
o simula e h ee-dimensional agg ega ed pa icle gel ne wo ks. The model
neglec s speci ic e ec s o changes in in amolecula in e ac ions du ing o
a e adso p ion, whils accoun ing o associa i e in e molecula in e ac ions
be ween adso bed pa icles h ough he o ma ion o s ong lexible bonds.
Con inemen o he pa icles o he in e ace is achie ed wi h a s eep po en ial
well o ini e wid h in he z-di ec ion (pe pendicula o he in e ace). Al hough
pa icles a e s ongly adso bed, hey s ill ha e some eedom o dis ibu e
hemsel es a ound hei equilib ium posi ions a Z=0. Fo his eason, he
Backg ound. P oduc s om p o eins
99
model o he laye is no comple ely wo-dimensional (Wijmans and Dickinson
1998).
Compe i i e adso p ion
Some o he complexi y o p o ein abso p ion is pa ly due o he
po en ial o compe i i e adso p ion be ween p o eins which a e p esen .
In mix u es in ol ing globula p o eins, he i e e sibili y o he
adso p ion e en s p e en s equilib ium om e e being achie ed. Al hough
mo e easily dena u able (“so ”) p o eins a e expec ed o ha e g ea e su ace
a ini y and exchangeabili y han mo e s able (“ha d”) p o eins, a use ul ule
o humb is ha he adso bed laye will be domina ed by he p o ein ha
p esen s i sel i s o he in e ace.
In cases whe e bo h p o eins a e p esen ed oge he in oughly equal
amoun s, he ex en o dominance o indi idual componen s will depend on
he esidence imes o indi idual p o ein molecules a he in e ace in ela ion
o hei a es o un olding. Thus, he compe i ion o occupy si es a he
in e ace may p e en ‘ea ly’ adso bed species om e e achie ing he
‘i e e sible s a e’, in which case an ini ially adso bing molecule may be
displaced.
The ini eness o he ime equi ed o achie e he i e e sible s a e is a
key poin because i allows o mul iple collisions o po en ially compe ing
molecules o ake place be o e any chance o eplacemen becomes
impossible.
The p o ein in e acial composi ion a any gi en bulk p o ein composi ion
a io appea s o be a ec ed la gely by he ela i e a es o a i al o he
componen s a he in e ace and he molecula a eas a ailable o hem a he
ime o a i al. In his sense he accumula ion o p o ein molecules a he luid-
luid in e ace om he mixed solu ion is no a he modynamically-con olled
Backg ound. P oduc s om p o eins
100
compe i i e adso p ion p ocess. This means ha he composi ion o he
in e ace canno be p edic ed om any simple he modynamic model
When small-molecule su ac an s a e p esen in p o ein-con aining
sys ems, he adso p ion o he p o ein is a ec ed by he binding o su ac an
o bo h he p o ein and he luid in e ace In addi ion o he p e e en ial
binding o su ac an o he su ace a high bulk concen a ions, su ac an
binding o hyd ophobic si es on he p o ein may also educe i s su ace
a ini y. The e o e he p o ein can be emo ed om he in e ace as a
consequence o wo dis inc mechanisms:
1) The solubilisa ion mechanism: deso p ion o p o ein a ises as a esul
o solubilisa ion in o he aqueous phase in he o m o a p o ein-
su ac an complex.
2) The eplacemen mechanism: displacemen o p o ein a ises because
su ac an lowe s he in e acial ee ene gy mo e e ec i ely han
does p o ein (o p o ein-su ac an complex).
Ionic su ac an s usually bind s ongly o p o eins, such ha compe i i e
adso p ion in ol ing cha ged amphiphiles can be ega ded as p oceeding
mainly by he solubilisa ion mechanism. Wi h mo e weakly in e ac ing non-
ionic su ac an s, howe e , he eplacemen mechanism can be ega ded as
(Wijmans and Dickinson 1998).
In e acial Measu emen s
Su ace p essu e-a ea
The Langmui adso p ion model is he mos common model used o
quan i y he amoun o amphiphilic compounds adso bed. This abso p ion is
a unc ion o pa ial p essu e o concen a ion and empe a u e. Fo he sake
o simplici y, his model conside s he adso p ion o an ideal gas on o an
Backg ound. P oduc s om p o eins
101
idealized su ace. Thus, when a monolaye is ab ica ed a he gas-liquid o
liquid-liquid in e ace, he ilm is named Langmui ilm (To h 2002).
A use ul me hod o cha ac e ise he Langmui ilm is by ob aining he
su ace p essu e/molecula a ea iso he ms, measu ed by he Langmui
me hod. The measu emen sys em consis s o a pla e i ed wi h wo mobile
ba ie s, and a de ice which is able o measu e su ace ension (e.g. Wilhelmy
pla e). Figu e 3.3-2 illus a es he de ice measu emen :
Figu e 3.3-2: Langmui ough de ice
Thus, he expe imen consis s o measu ing he su ace ension as a
unc ion o he mean molecula a ea pe pendicula on he monolaye as is
comp essed by he wo ba ie s.
Figu e 3.3-3 shows a ypical p essu e-molecula a ea cu e o an
amphiphilic monolaye adso bed a he in e ace ob ained when he ba ie s
a e mo ed owa ds each o he . The di e en phases occu ing o e he
adso p ion iso he m a e also ep esen ed in his igu e.
A he beginning, when he ba ie s a e a hei g ea es dis ance om
each o he , he monolaye is in he gaseous phase whe e he hyd ophobic
g oups a e comple ely sepa a ed. Fu he comp ession ( om igh o le )
o ces he monolaye molecules in o he liquid phase ha causes a sligh
ele a ion o he su ace p essu e, s a ing wi h he so-called “li -o ” poin .
Fu he comp ession squeezes he amphiphilic molecules in o a solid, which

Backg ound. P oduc s om p o eins
102
gi es a s eep ise in he su ace ension. By u he inc easing he comp ession,
he laye collapses in o a many-laye ed s uc u e (To h 2002).
Figu e 3.3-3: Su ace p essu e Vs. Molecula a ea o an amphiphilic molecule
Dila a ional D ople Tension
When a d op o a luid is in con ac wi h o he luid, he d op exhibi s a
combina ion o e ec s coming om he su ace ension (which ends o o m
a sphe ical d op) and he g a i a ional o ce (which ends o elonga e he
d op). In his way, he asymme ic d op can be exp essed by using he Laplace
equa ion:
1
𝑥𝑑(𝑥𝑠𝑒𝑛𝜃)
𝑑𝑥 =2
𝑏−𝑐𝑧
(3.3-4)
Whe e x and z a e he ca esian coo dina es, b is he adius o cu a u e,
Ѳ is he angen angle and c is a cons an ela ed o he Capila numbe , which
is de ined as : c=g·Δρ/γ, whe e g is he g a i y accele a ion, Δρ is he di e ence
o densi y be ween bo h s udied luids and γ he su ace ension.
Mean molecule a ea [Å2]
Su ace p essu e [mN/m]
Gaseous
Liquid
Solid
Collapse
Backg ound. P oduc s om p o eins
103
In e acial Shea Rheology
Two di e en kinds o in e acial heological measu emen s can be
dis inguished by applying shea o dila a ional de o ma ions o a luid- luid
in e ace.
When he in e ace is shea ed, bo h he a ea and he amoun o
su ac an in he in e ace emain cons an and hen i is possible o measu e
he equi ed shea o ce applied o he plane o he in e ace. Fo mos
sys ems, shea a e hinning occu s and he obse ed iscosi y is an appa en
in e acial iscosi y. Fo ins ance, alues o globula p o eins usually show a
high expe imen al unce ain y, since he monolaye can yield o up u e and
he measu ed “ iscosi y” will g ea ly depend on he up u e pa e n.
On he o he hand, i he in e acial a ea is enla ged by dila a ional
de o ma ion, lea ing i s shape unal e ed, an inc ease in in e acial ension
akes place, because he mola amoun o adso bed ma e ial pe uni su ace
a ea (su ace load, Γ) is dec eased. This is usually exp essed in he su ace
dila a ional modulus, de ined as ollows:
𝐸𝑆𝐷=𝑑𝛾
𝑑ln𝐴
(3.3-5)
whe e A is he su ace a ea.
Fo p o eins ESD may be la ge and less dependen on ime, because
p o eins adso b mo e o less i e e sibly. Howe e , he concen a ion o
p o ein a he in e ace has a la ge e ec . In addi ion, changes in p o ein
con o ma ion upon adso p ion and dila ion can also a ec he modulus.
Su ace heological pa ame e s o p o ein laye s depend on pH, ionic
s eng h, sol en quali y, empe a u e. Howe e , moduli and iscosi ies a e
usually a hei maximum alues nea he isoelec ic pH. In any case, he
measu emen o ESD is di icul and so is he in e p e a ion o he esul s
Backg ound. P oduc s om p o eins
104
ob ained om dila a ional heological measu emen s (Damoda an, Pa kin e
al. 2007).
Backg ound. P oduc s om p o eins
105
3.4. P oduc s om p o ein
Emulsions
Emulsions a e dispe se sys ems consis ing o wo immiscible liquids. One
o he phases (called he in e nal o dispe se phase) is dispe sed in he o m
o small d ople s in a liquid medium (called he con inuous phase). Acco ding
o he hyd ophobici y, some classes may be dis inguished: oil-in-wa e (O/W),
wa e -in-oil (W/O), and oil-in-oil (O/O). To dispe se wo immiscible liquids, i
is needed a hi d componen , namely, he emulsi ie . The choice o he
emulsi ie is c ucial in he o ma ion o he emulsion and i s long- e m s abili y
(Tad os 2013).
Classi ica ion acco ding o sys em s uc u e:
1. O/W and W/O mac oemulsions: D ople size ange o 0.1–5 μm wi h an
a e age o 1–2 μm. They a e kine ically s able.
2. Nanoemulsions: D ople size ange o 20–100 nm. Simila o
mac oemulsions, hey a e only kine ically s able.
3. Micella emulsions o mic oemulsions: hese usually ha e he size ange
o 5–50 nm. They a e he modynamically s able.
4. Double and mul iple emulsions: hese a e emulsions-o -emulsions,
W/O/W, and O/W/O sys ems.
5. Mixed emulsions: hese a e sys ems consis ing o wo di e en dispe se
d ople s ha do no mix in a con inuous medium.
Me hods o Emulsi ica ion
Se e al p ocedu es may be applied o emulsion p epa a ion, including:
simple pipe low de ices (low agi a ion ene gy); s a ic mixe s and gene al
Backg ound. P oduc s om p o eins
112
1) Use o mixed su ac an ilms: In many cases using mixed su ac an s
can educe coalescence
2) Fo ma ion o lamella liquid c ys alline phases a he O/W in e ace:
As a esul o mul ilaye s uc u es, he po en ial d op is shi ed o longe
dis ances hus educing he an de Waals a ac ion.
Food emulsions
E e y hing abo e desc ibed abou emulsions is applicable o ood
emulsions, he only di e ence is ha now he sys em is e y complex, and
p o eins a e usually used as emulsi ie s ins ead o su ac an s.
Thus, a simila bulk concen a ions (w/ ), low molecula mass
su ac an s dec ease he su ace ension o a g ea e ex en han he
mac omolecula su ac an s. This di e ence is mainly ela ed o di e ences in
o ien a ion and con igu a ion o hese su ac an s a an in e ace. Al hough
low molecula mass su ac an s a e mo e e ec i e han p o eins in educing
he in e acial ension, su ac an -based oams and emulsions a e gene ally
mo e uns able han hose p ocessed using p o eins.
This is because p o eins, in addi ion o lowe ing in e acial ension, can
o m a con inuous iscoelas ic memb ane-like ilm a ound oil d ople s o ai
cells ia non-co alen in e molecula in e ac ions and ia co alen disulphide
c oss-linking. Consequen ly, in oods, which con ain bo h low molecula and
mac omolecula su ac an s, he s abili y o colloidally dispe sed phases is
p ima ily dependen on p o ein ilms adso bed a he in e aces. Howe e ,
p ac ical obse a ions indica e ha all p o eins a e no equally su ace ac i e,
e en hough all a e amphiphilic and a majo i y o hem con ain simila
pe cen ages o pola and nonpola amino acid esidues (Tad os 2013).

Backg ound. P oduc s om p o eins
113
The di e ences ound in he su ace ac i i ies o a ious p o eins mus be
ela ed o di e ences in hei con o ma ion and he suscep ibili y o hose
con o ma ions o un old a in e aces. In ui i ely, he molecula ac o s ha
in luence su ace ac i i y o p o eins mus be ela ed o lexibili y,
con o ma ional s abili y a in e aces, apid adap abili y o he con o ma ion
o changes in i s en i onmen , and o he dis ibu ion pa e n o hyd ophilic
and hyd ophobic esidues in i s p ima y s uc u e, as well as on i s olded
su ace.
In addi ion, apa om he in insic molecula ac o s, he su ace ac i i y
o a p o ein will be also dic a ed by se e al ex insic ac o s such as pH, ionic
s eng h, and empe a u e (Damoda an 1997).
Emulsion cha ac e isa ion
Rheology o emulsions
Emulsions gene ally show a heological beha iou i ing in o he
ca ego y o complex luids. Howe e , hey di e om o he complex luids in
h ee main aspec s:
1) The mobile liquid/liquid in e ace ha con ains su ac an o polyme
laye s.
2) The dispe sed-phase iscosi y ela i e o ha o he medium has an
e ec on he heology o he emulsion.
3) The de o mable na u e o he dispe sed-phase d ople s.
Consequen ly, we can di e ence be ween in e acial and bulk heology.
Backg ound. P oduc s om p o eins
114
In e acial Rheology
Mixed Su ac an Films
Acco ding o he da a a ailable, he e seems o be a ela ionship be ween
he use o a mix u e o su ac an s and he enhancemen o he s abili y o he
emulsion. This could be due o he inc ease o in e acial dila a ional elas ici y
ESD o he mixed ilm when compa ed o ha one con aining a single
su ac an . Howe e , o he ac o s such as hinning o he ilm be ween
emulsion d ople s can also play a majo ole.
P o ein Films
The iscoelas ic p ope ies o p o ein ilms a he O/W in e ace also
co ela es well wi h he s abili y o emulsion d ops agains coalescence.
Some iscoelas ic measu emen s can be ca ied ou using c eep- eco e y
es s. The s abili y o he emulsion was assessed by measu ing he esidence
ime o se e al oil d ople s a a plana O/W in e ace con aining he adso bed
p o ein. Thus, Biswas and Haydon (1963) de i ed a ela ionship be ween
coalescence ime (τ) and in e acial pa ame e s such as su ace iscosi y (ηs),
ins an aneous modulus (Go), and adso bed ilm hickness (h) (EQ. 3.4-3).
𝜏=𝜂𝑠∙[3𝐶′ℎ2
𝐴−1
𝐺0−𝜙(𝑡)]
(3.4-3)
whe e 3C’ is a c i ical de o ma ion ac o , A is he Hamake cons an (Van
de Waals body-body in e ac ion) and φ( ) is he elas ic de o ma ion pe uni
s ess.
The equa ion analysis shows ha iscoelas ici y is necessa y (eg. τ
inc eases wi h inc easing ηs) bu no su icien o ensu e s abili y agains
coalescence. Film hickness seems o be he mos impo an ac o , such ha
o ensu e s abili y o an emulsion h mus be la ge enough.
Backg ound. P oduc s om p o eins
115
Bulk heology
Dilu ed emulsions
Fo highly iscous oil d ople s dispe sed in low iscosi y media such as
wa e , dilu e O/W emulsions ( olume ac ion φ ≤ 0.01) o non-in e ac ing
d ople s beha e as ‘‘ha d sphe es’’. In his case, he ela i e iscosi y (η ) is
gi en by he Eins ein equa ion (3.4-4):
𝜂𝑟=1+[η]ϕ
(3.4-4)
whe e [η] is he in insic iscosi y (2.5 o ha d sphe es).
Fo d ople s wi h a iscosi y compa able o ha o he medium, he
ansmission o angen ial s ess ac oss he O/W in e ace, om he
con inuous phase o he dispe sed phase, causes liquid ci cula ion inside he
d ople s. Ene gy dissipa ion is less han ha o ha d sphe es and he ela i e
iscosi y is lowe han ha p edic ed by he Eins ein equa ion.
Thus, o an emulsion wi h iscosi y ηi o he dispe se phase and ηo o
he con inuous phase, he in insic iscosi y can be de ined as show EQ.
(3.4-5).
𝜂=2.5 (𝜂𝑖+0.4𝜂0
𝜂𝑖+𝜂0)
(3.4-5)
When ηi >> ηo, he d ople s beha e as igid sphe es and η app oaches he
limi alue o 2.5. In con as i ηi << ηo (e.g. in oams), [η] = 1.
I he olume ac ion o d ople s exceeds he Eins ein limi , (φ >0.01), i
mus ake in o accoun he e ec o B ownian mo ion and in e pa icle
in e ac ions.
The smalle he emulsion d ople s, he mo e impo an he con ibu ion
o B ownian mo ion and colloidal in e ac ions. B ownian di usion ends o
andomize he posi ion o colloidal pa icles, leading o he o ma ion o
Backg ound. P oduc s om p o eins
116
empo a y double s, iple s, and so on. The hyd odynamic in e ac ions a e o
longe ange han he colloidal in e ac ions, and hey come in o play a
ela i ely low olume ac ions (φ >0.01) esul ing in o de ing o he pa icles
in o laye s and ending o des oy he empo a y agg ega es caused by he
B ownian di usion. This explains he shea hinning beha iou o emulsions a
high shea a es.
Fo he olume ac ion ange 0.01 < φ < 0.2, Bachelo (1977) de i ed he
ollowing exp ession o a dispe sion o hyd odynamically in e ac ing ha d
sphe es:
𝜂𝑟=1+2.5𝜙+6.2𝜙2+𝜐𝜙3
(3.4-6)
Whe e he i s pa co esponds o he Eins ein limi (EQ. 3.4-4) while
he hi d e m accoun s o hyd odynamic ( wo-body) in e ac ions and he
ou h e m ela es o mul ibody in e ac ions.
Concen a ed emulsions
Conside ing he heology o concen a ed emulsions, an exp ession o
he ou h e m in φ3 o EQ. (3.4-8) should be p o ided. Un o una ely, he e
is no heo e ical igo ous ea men o his e m and only semiempi ical
equa ions o in e media e olume ac ions a e a ailable (Phan-Thien and
Tanne 1999, Pal 2000).
Two models we e p oposed by Pal (2001):
𝜂𝑟[2𝜂𝑟+5𝜆
2+5𝜆]1/2=𝑒[2.5𝜑
1−𝜑
𝜑∗]
(3.4-7)
𝜂𝑟[2𝜂𝑟+5𝜆
2+5𝜆]1/2=[1−𝜑/𝜑∗]2.5𝜑∗
(3.4-8)
Backg ound. P oduc s om p o eins
117
whe e λ is he a io o iscosi ies o dispe se d ops and con inuous
medium and φ* is he limi o closes packing o d ops in ee space.
An inc ease in he concen a ion o d ops in emulsions esul s no only in
an inc ease in iscosi y a low shea a es bu also in he appea ance o s ong
non-New onian e ec s leading o a shea a e dependence o he appa en
iscosi y. A ema kable ansi ion om an almos New onian beha iou a low
s esses o an anomalous low wi h p onounced non-New onian e ec s may
also akes place (Tad os 2013).
Mic oscopy
The human eye is able o esol e objec s ha a e g ea e han 0.1 mm.
Many o he s uc u al componen s in ood emulsions (such as d ople s,
su ac an micelles, a c ys als, gas bubbles o p o ein agg ega es) a e smalle
han his limi and canno be dis inguished by humans.
Se e al echniques a e a ailable o p o ide in o ma ion abou he
s uc u e, dimensions, and o ganiza ion o he componen s wi hin ood
emulsions, o example, op ical mic oscopy, elec on mic oscopy (SEM) o
a omic o ce mic oscopy (AFM). These echniques ha e he abili y o p o ide
ele an in o ma ion abou complex sys ems in he o m o “images”
(McClemen s 2004).
Con en ional op ical mic oscopy
The op ical mic oscope is one o he mos aluable ools o obse ing he
mic os uc u e o emulsions. The op ical mic oscope con ains se e al lenses
ha di ec ligh h ough he specimen and magni y he esul ing image. The
esolu ion is de e mined by he wa eleng h o ligh used and he mechanical
design o he ins umen , being he heo e ical limi o esolu ion o an op ical
mic oscope abou 0.2 μm. Ne e heless, i p o ides use ul in o ma ion abou
he size dis ibu ion o d ople s in emulsions ha con ain la ge d ople s, and

Backg ound. P oduc s om p o eins
118
can o en be used o dis inguish be ween loccula ion and coalescence, which
is some imes di icul using ins umen al pa icle sizing echniques based on
ligh sca e ing (McClemen s 2004)
Lase Scanning Con ocal Mic oscopy
Lase Scanning Con ocal Mic oscopy (LSCM) can p o ide ex emely use ul
in o ma ion abou he mic os uc u e o ood emulsions. This echnique has
he ad an age ha p o ides highe cla i y images han con en ional op ical
mic oscopy, and o en allows he gene a ion o h ee-dimensional images o
s uc u es wi hou he need o physically sec ioning he specimen. The LSCM
ocuses an ex emely na ow lase beam a a pa icula poin in he specimen
being analysed and a de ec o measu es he in ensi y o he esul ing
luo escence signal. 3D images can be ob ained by ocusing he lase beam a
di e en e ical dep hs. The obse a ion o he mic os uc u e o
mul icomponen sys ems is o en acili a ed by using he na u al luo escence
o ce ain componen s (such as p o eins) o by using luo escen dyes
(McClemen s 2004).
Scanning Elec on Mic oscopy
Scanning Elec on Mic oscopy (SEM) is widely used o examine he
mic os uc u e o ood emulsions, especially hose ha con ain s uc u al
componen s ha a e smalle han he lowe limi o esolu ion o op ical
mic oscopes. This echnique can p o ide ele an in o ma ion abou he
concen a ion, dimensions, and spa ial dis ibu ion, whe eas mic os uc u e is
no signi ican ly al e ed by he sample
Elec on mic oscopes use elec on beams, ins ead o ligh beams, o
p o ide in o ma ion abou he s uc u e o ma e ials. These beams a e
di ec ed h ough he mic oscope using a se ies o magne ic ields, ins ead o
op ical lenses, which used op ical mic oscope. Elec on beams ha e much
Backg ound. P oduc s om p o eins
119
smalle wa eleng hs han ligh and so hey can be used o examine much
smalle objec s (abou 0.2 nm)
S a ic ligh sca e ing
D ople size analysis ins umen s ha use s a ic ligh sca e ing (also
called lase di ac ion) a e based on he p inciple ha a beam o ligh is
di ec ed h ough an emulsion, he lase is sca e ed by he d ople s in a well-
de ined manne . A measu emen o he ex en o ligh sca e ing by an
emulsion can be used o de e mine he d ople size dis ibu ion and
concen a ion by using a ma hema ical model o ela e he measu ed da a o
he pa icle cha ac e is ics.
Food Gels
De ini ion
A ood gel can be conside ed as a high mois u e h ee-dimensional
polyme ic ne wo k ha esis s low unde p essu e and is able o e ain hei
dis inc s uc u al shape. Gene ally, a gel is a con inuous ne wo k o
in e connec ed pa icles o asso ed mac omolecules dispe sed in a
con inuous liquid phase. The gela ion is he phenomenon which in ol es he
associa ion o c osslinking o he polyme chains o o m a h ee-dimensional
ne wo k ha immobilizes wa e wi hin i .
Food hyd ocolloids a e usually he mos equen ly used gelling agen s in
ood p oduc s. A wide ange o polysaccha ides and p o eins a e nowadays
a ailable as ood hyd ocolloids de i ed om na u al sou ces. Polysaccha ides
a e inco po a ed because o hei abili y o con ol s abili y and ex u e o
oods, as well as o hei ole in encapsula ion and con olled elease o ac i e
agen s ( la ou s, unc ional ing edien s, e c.) (Bane jee and Bha acha ya
2012).
Backg ound. P oduc s om p o eins
120
In ood gels he liquid is in a iably wa e and he molecula ne wo k
consis s o p o eins o polysaccha ides o a combina ion o bo h. The
p ope ies o he gel a e he ne esul s o he complex in e ac ions be ween
he wa e and he molecula ne wo k. The wa e , as a sol en , has in luence
in he na u e and magni ude o he in e molecula o ces ha main ain he
in eg i y o he polyme ne wo k. The polyme ne wo k holds he wa e ,
p e en ing i om lowing away.
Un o una ely he complexi y o hese in e ac ions, esponsible o he
use ul unc ional p ope ies o gels, makes i e y di icul o p edic
quan i a i ely hei physical p ope ies, e en o pu e p o eins o
polysaccha ides. In addi ion, ood hyd ocolloids a e a ely pu e and a e o en
used wi h o he ing edien s ha inc ease he deg ee o complexi y o ood
sys ems. Fo his eason, hei physical p ope ies mus gene ally be ea ed
empi ically (Damoda an 1997).
P o ein gela ion
Hea --induced gela ion, pH-induced gela ion o high p essu e gela ion a e
echniques widely used o he gela ion o globula p o eins and p oceed
h ough a se ies o ansi ions, such as dena u a ion (un olding) o na i e
p o eins, agg ega ion o un olded molecules, s and o ma ion om
agg ega es, and associa ion o s ands in o a ne wo k (Bane jee and
Bha acha ya 2012).
Hea -induced gela ion
Hea -induced gela ion is p obably he mos impo an and common
me hod o ob ain gels. Gela ion in ol es di e en s eps om p o ein
dena u a ion o o ma ion o p o ein agg ega es and associa ion o agg ega es
o o m a h ee-dimensional ne wo k:
Backg ound. P oduc s om p o eins
121
- The i s s ep always consis s o p o ein dena u a ion in ol ing
un olding (a leas pa ially) o dissocia ion o he molecules induced
by he mal ene gy. As a consequence, some o he hyd ophobic
g oups, which emained bu ied in he p o ein co e unde na i e
con igu a ion, become exposed o he aqueous phase abo e some
empe a u e.
- The second s ep akes place h ough hyd ophobically d i en p o ein-
p o ein in e ac ions ha lead o he associa ion and agg ega ion o
un olded molecules o o m complexes o highe molecula weigh . In
his s age, disulphide (-S-S-) bonds may also play an impo an ole in
combina ion wi h hyd ophobic in e ac ions.
- Random associa ion o agg ega es o o m a h ee-dimensional
s uc u e which ex ends o he whole sys em has been sugges ed as a
hi d s ep. Meanwhile he con inuous phase is en apped wi hin he
ne wo k (Cla k, Ka anagh e al. 2001).
The eac ion a e can be ypically de e mined ei he by he un olding o
by he agg ega ion eac ion, depending on he a io o he eac ion a es o
he single s eps (Bane jee and Bha acha ya 2012). Se e al a iables,
depending ei he on p o ein na u e and composi ion o on en i onmen al
ac o s, such as pH o ionic s eng h may exe an impo an in luence on
p o ein-p o ein and p o ein-sol en in e ac ions and as a esul on hese
eac ion a es, hus condi ioning he ype o gel ne wo k o med.
Essen ially, p o eins can agg ega e in wo ways. One is by andom
agg ega ion which can lead o he e ogeneous pa icula e ne wo k s uc u es.
The o he is by linea agg ega ion ha gi es ise o ine s anded (s ing o
beads) ne wo k s uc u es. Many p o eins (e.g. globula p o eins) can o m
ei he ype o gel ne wo k depending on he balance o o ces. The
Backg ound. P oduc s om p o eins
128
In small-s ain es ing he s ain mus be low enough o a oid any
un eco e able s uc u al change (e.g. SAOS es s). On he o he hand, la ge-
s ain es ing e e s o de o ming a sample abo e o he poin o pe manen
s uc u al change. This la e g oup o es s o en yields in o ma ion ha
co ela es wi h senso y e alua ion (Sch amm 2000).
Small-s ain es ing. Oscilla o y es
Since gels exhibi iscoelas ic beha iou , dynamic heological es s o
e alua e p ope ies o gel sys ems a e widely used o s udying he
cha ac e is ics o gels as well as gela ion and mel ing. Figu e 3.4-2 illus a es
he change in modulus when he gel ans o ms i s liquid-like s uc u e o gel-
like s uc u e.
Figu e 3.4-2: Viscoelas ic esponse o a ma e ial
I G’ is much g ea e han G’’, he ma e ial will beha e mo e like a solid;
ha is, he de o ma ions will be essen ially elas ic. Howe e , i G’’ is much
g ea e han G’, he ene gy used o de o m he ma e ial is dissipa ed iscously
and he ma e ial exhibi s liquid-like beha iou .
Th ee ypes o dynamic es s a e usually used o ob ain use ul p ope ies
o gels, gela ion, and mel ing:

Backg ound. P oduc s om p o eins
129
1) F equency sweep s udies in which G’ and G’’ a e de e mined as a
unc ion o equency, a ixed empe a u es.
2) Tempe a u e sweep es s in which G’ and G’’ a e de e mined as a
unc ion o empe a u e a ixed equency.
3) Time sweep in which G’ and G’’ a e de e mined as a unc ion o ime
a ixed equency and empe a u e.
Kine ics o Gela ion
Measu emen o gela ion kine ics equi es a me hod o ollowing he
de elopmen o he gel ne wo k wi hou signi ican ly a ec ing he p ocess by
mechanical dis u bance.
The mos widely used echnique o s udy gela ion kine ics, a isen a e
he de elopmen o con olled-s ess heome e s, in ol es o ma ion o he
gel be ween he pla es o an oscilla o y heome e (He mansson 1986). Wi h
his ype o ins umen , p o ided ha condi ions a e chosen app op ia ely, i
is possible o moni o he e olu ion o he linea iscoelas ic unc ions o e
he gela ion p ocess (Damoda an 1997).
In ac , nowadays he mos uni e saly accep ed de ini ion o he gel poin
comes om he de e mina ion o linea iscoelas ici y p ope ies associa ed
o he de elopmen o a sel -simila s uc u e o he so-called c i ical gel
(Win e 1987). Ma e ials a he gel poin exhibi a dis inc heological
beha iou whe e he ollowing powe law equa ion holds o linea
iscoelas ic unc ions:
𝐺′(𝜔)=𝑘1·𝜔𝑛
(3.4-9)
𝐺′′(𝜔)=𝑘2·𝜔𝑛
(3.4-10)
Backg ound. P oduc s om p o eins
130
whe e n is he elaxa ion exponen and k1, k2 a e cons an s ela ed o his
exponen and o he gel s i ness. As a consequence, he loss angen is
equency independen a he c i ical gel. In p ac ice, he gel poin has been
widely de e mined as he poin a which an(δ) is no a unc ion o equency.
Gel S eng h
Many di e en ins umen s a e a ailable o measu ing gel s eng h.
Some simply esul s ha p o ide empi ical es s canno be ela ed o
undamen al heological quan i ies. Howe e , he e a e o he es s ha can
measu e well-de ined pa ame e s such as shea modulus o up u e s eng h.
A ypical es measu es he o ce applied o he gel e sus displacemen .
In any case, as p e iously men ioned, mode n heome e s a e he mos
ex ended de ices used o cha ac e ize gels. Mo eo e , among he di e en
measu emen echniques ha heome e s can pe o m, SAOS measu emen s
o iscoelas ic p ope ies using pa allel pla es o cone-pla e geome ies a e
p e e ed, since hey allow moni o ing he change in s uc u e o e gela ion,
which is based on he assump ion ha he ma e ial's beha iou is linea . Gel
s eng h can hen be ela ed o linea iscoelas ic unc ions (G', G'', an δ, e c).
Wa e holding capaci y (WHC)
Mainly, he e a e wo causes which con ibu e o he wa e holding
capaci y o a ma e ial: he pola i y (including su ace cha ges) and he
capilla i y, which is he mos impo an . Fo his unc ional p ope y,
myo ib illa p o eins a e he mos impo an . Thus, wa e is able o be held
mainly in he holes be ween ac in and myosin ilamen s.
Mic oscopy
Mic oscopy echniques a e widely used o he cha ac e isa ion o he
p o ein mic os uc u e. Op ical mic oscopy may be used, howe e LSCM and
Backg ound. P oduc s om p o eins
131
SEM, desc ibed abo e, a e some o he mos use ul echniques o he
isualiza ion o gel mic os uc u e.
Nu i ional p ope ies
An ioxidan p ope ies o ood gels
F ee adicals a e gene a ed h ough no mal eac ions wi hin he body
du ing espi a ion in ae obic o ganisms. The p esence o hese po en ially
oxic p oduc s can gi e ise o se e al diseases. Ai pollu an s and oxidan s in
obacco can ypically cause some ha m ul eac ions in skin o can be abso bed
o blood ci cula ion, exe ing some ad e se e ec s. Addi ionally, UV adia ion
can be a p oduce o a a ie y o oxidan s.
The ee adicals, which a e physiologically p oduced, can p o ide a
p o ec ion agains in ec ions. Acco ding o he ee adical heo y o ageing
de eloped by Denham Ha man, o ganisms age when ee adicals accumula e
in cells and cause ha m o e ime. In his way, eac i e species can cause
damage in p o eins, and mu a ions in DNA, oxida ion o memb ane
phospholipids and modi ica ion in low densi y lipop o eins (LDL). Thus,
an ioxidan compounds can emo e eac i e species h ough enzyma ic and
non-enzyma ic an ioxidan s. Howe e , in ce ain ci cums ances he
endogenous immune sys em ails o p o ec he body agains eac i e adicals
on i s own.
This b ings abou he need o syn he ic and na u al an ioxidan s, which
can p e en oxida i e s ess and i s dele e ious e ec s. Syn he ic an ioxidan s
a e cos -e ec i e and e icien bu display some oxic and haza dous e ec s.
In he a eas o human nu i ion, and biochemis y, na u al an ioxidan s om
ood esou ces ha e been he ocus o g owing in e es o hei po en ial
heal h bene i s wi h li le o no side e ec s (Sa madi and Ismail 2010).
Backg ound. P oduc s om p o eins
132
An ioxida i e pep ides in ood p oduc s
Se e al pep ides om p o ein ing edien s ha e been ound o possess
an ioxidan abili y. An ioxidan pep ides om oods con ain 5-16 amino acid
esidues and a e conside ed o be sa e and heal hy compounds wi h low
molecula weigh , low cos , high ac i i y and easy abso p ion. They ha e some
ad an ages in compa ison o enzyma ic an ioxidan s; ha is, wi h simple
s uc u e hey ha e mo e s abili y in di e en si ua ion and no haza dous
immuno eac ion. In addi ion, hey p esen nu i ional and unc ional
p ope ies beside hei an ioxidan ac i i y. The e a e wo di e en o ms o
an ioxidan ac i i y: ei he as hyd olysa es o p ecu so p o eins o as
bioac i e pep ides. Hyd olysa e is a mix u e ha is mainly composed o
pep ides and amino acids which a e p oduced h ough p o ein hyd olysis by
enzyme, acid o alkali ea men
The exac mechanism unde lying he an ioxidan ac i i y o pep ides has
no ully been unde s ood, ye a ious s udies ha e displayed ha hey a e
inhibi o s o lipid pe oxida ion, sca enge s o ee adicals and chela o s o
ansi ion me al ions. An ioxida i e p ope ies o he pep ides a e mo e
ela ed o hei composi ion, s uc u e, and hyd ophobici y. Ty , T p, Me , Lys,
Cys, and His a e examples o amino acids ha cause an ioxidan ac i i y.
Amino acids wi h a oma ic esidues can dona e p o ons o elec on de icien
adicals. This p ope y imp o es he adical-sca enging p ope ies o he
amino acid esidues. I is p oposed ha he an ioxida i e ac i i y o His-
con aining pep ides is in ela ion wi h he hyd ogen-dona ing, lipid pe oxyl
adical apping and/o he me al ion-chela ing abili y o he imidazole g oup.
In addi ion, SH g oup in cys eine has an independen ly c ucial an ioxidan
ac i i y due o i s di ec in e ac ion wi h adicals. Ne e heless, p o ein linkage
con o ma ion and s uc u al ea u es o he pep ides ha e been claimed o
in luence an ioxidan abili y (Sa madi and Ismail 2010).
Backg ound. P oduc s om p o eins
133
Bioplas ics
The bioplas ics indus y is a s ongly g owing pa o he plas ics indus y.
Wi h a global p oduc ion capaci y a ound 1.7 million onnes pe yea in 2014,
he olume in he ma ke is s ill small compa ed o he o e all plas ics olume
o 320 million onnes/yea . Howe e , acco ding o ecen es ima ions, he
sha e o bioplas ics will inc ease up o 7.8 million onnes o bioplas ics (2-3
pe cen o plas ics ma ke ) in 2019 (Plas ics-Eu ope 2008).
Acco ding o Eu opean-bioplas ic Associa ion, he e m bioplas ic
encompasses a whole amily o ma e ials which ha e some di e ences wi h
con en ional plas ics because hey a e bio-based, biodeg adable, o bo h.
Thus, wo kinds o bioplas ic ma e ials can be dis inguish acco ding o his
de ini ion:
- Bio-based ma e ials, in which he ma e ial o p oduc is de i ed om
biomass, which can s em om ei he plan o animal sou ces.
- Biodeg adable polyme ic ma e ials, whe e he e m biodeg adable
e e s o a chemical p ocess du ing which mic o-o ganisms, which a e
a ailable in he en i onmen , con e ma e ials in o na u al
subs ances. Ob iously, he p ocess o biodeg ada ion depends on he
su ounding en i onmen al condi ions (mois u e, empe a u e, ligh ,
O2), and on he ma e ial i sel . Biodeg adabili y is an inhe en p ope y
o ce ain bioplas ic ma e ials ha can bene i speci ic applica ions
(Plas ics-Eu ope 2008)
Ma e ials used in he bioplas ic manu ac u ing
Usually, a bioplas ic ma e ial (o he biodeg adable g oup) consis s o a
polyme ic ne wo k o med by a biodeg adable mac omolecula subs ance (in
his case is a p o ein) and a plas icise .

Backg ound. P oduc s om p o eins
134
Na u al aw-ma e ials
In ecen yea s he e has been a g ea in e es o u ilize enewable
biomass in o de o manu ac u e consume goods which exhibi high-quali y,
cos -compe i i e and biodeg adable, educing he consump ion and he
dependence on pe ochemical eeds ock and diminishing en i onmen al
pollu ion (Rosen a e and O ieno 2006, Felix, Ma in-Al onso e al. 2014).
Mainly, p o eins and polysaccha ides ha e been pos ula ed as enewable
biomass o manu ac u e biopolyme s o many yea s (De G aa 2000,
He nandez-Izquie do and K och a 2008). Polysaccha ides a e na u ally
ex ended, and a e widely used o ood indus y. These compounds ha e been
also used o bioplas ics (e.g. s a ch and chi osan a e good examples o
polysaccha ides used o his pu pose). As ega ds p o eins, hey a e a
enewable, biodeg adable esou ce wi h g ea po en ial o imp o e he
quali y and s abili y o a la ge ange o ood p oduc s by using a numbe o
p ocessing echniques (Rome o, Co dobes e al. 2008, Jayasunde a, Adhika i
e al. 2009, E ni, Windhab e al. 2011). Fo a long ime, p o eins ha e been
used o p oduce edible ma e ials, bu unde s anding o he p ecise physical
and chemical mechanisms o p o ein in e ac ions, hey can be used o p oduce
s able bioplas ic ma e ials (He nandez-Izquie do and K och a 2008, Balague ,
Gomez-Es aca e al. 2011).
Plas icise s
Plas icize s a e gene ally added o imp o e he p ocessabili y o he
p o ein ne wo k, as well as in o de o modi y he p ope ies o he inal
s uc u e, dec easing he glass ansi ion and he b i leness. Usually,
plas icize s consis o compounds which exhibi low-molecula weigh , low
ola ili y and ha in e ac wi h he polyme chains p oducing swelling
(He nandez-Izquie do and K och a 2008).
Backg ound. P oduc s om p o eins
135
This ype o compounds is widely used in polyme indus ies as addi i es.
The p ima y ole o such subs ances is o imp o e he lexibili y and
p ocessabili y o polyme s by lowe ing he second o de ansi ion
empe a u e, he glass ansi ion empe a u e (Tg). The council o he IUPAC
(In e na ional Union o Pu e and Applied Chemis y) de ined a plas icize as ‘‘a
subs ance o ma e ial inco po a ed in a ma e ial (usually a plas ic o
elas ome ) o inc ease i s lexibili y, wo kabili y, o dis ensibili y’’.
These subs ances educe he ension o de o ma ion, ha dness, densi y,
iscosi y and elec os a ic cha ge o a biopolyme , a he same ime as
inc easing he polyme chain lexibili y, esis ance o ac u e and dielec ic
cons an . O he p ope ies a e also a ec ed, such as deg ee o c ys allini y,
op ical cla i y, elec ic conduc i i y, i e beha iou and esis ance o biological
deg ada ion (Viei a, da Sil a e al. 2011).
The e a e many plas icize o p o ein-based bioplas ics, such as: 1,4-
Bu anediol, DATEMa, Dibu yl, Glyce ol, Lac ic acid, Oc anoic, Palmi ic acid,
So bi ol, Suc ose and Wa e (He nandez-Izquie do and K och a 2008).
Fo his s udy, Glyce ol has been he chosen plas icize . This is a widely
used bioplas ic, exhibi ing hyd ophilic p ope ies wi h a low molecula weigh ,
and high boiling poin . I s high plas icizing e ec has been a ibu ed o he
ease wi h which glyce ol can inse and posi ion i sel wi hin he 3-dimensional
biopolyme ne wo k (di Gioia and Guilbe 1999).
Me hods o p o ein-based bioplas ics
manu ac u ing
Gene al app oach
P o eins o e a la ge ange o possible physical and chemical
in e ac ions. This dual cha ac e is gi en because p o eins can pa icipa e in
non-co alen in e ac ions such as ionic, hyd ogen, and an de Waals bonding
Backg ound. P oduc s om p o eins
136
o in chemical eac ions h ough co alen linkage (pep ide and disulphide
bonds).
Usually, he o ma ion o he p o ein ne wo k is di ided in wo main
s ages: Plas iciza ion and p o ein in e ac ions (He nandez-Izquie do and
K och a 2008).
The plas icizing e ec o small pola molecules has been desc ibed in
e ms o inse ion and posi ioning wi hin he 3-dimensional p o ein ne wo k.
Mainly, he e a e ou heo ies o explain his p ocess:
1) The lub ici y heo y, whe e he plas icize is ac ing as a lub ican o
acili a e mobili y o he chain molecules.
2) The gel heo y, which conside s he dis up ion o polyme -polyme
in e ac ions (weak physical in e ac ions).
3) The ee olume heo y, which conside s ha he plas icize inc eases
he ee olume and mobili y o polyme chains (used o unde s and
he e ec o plas icize s in lowe ing he glass ansi ion empe a u e).
4) The coiled sp ing heo y, which explains plas icizing e ec s om he
poin o iew o angled-mac omolecules.
Howe e , he a ious possible ways in which p o eins may in e ac du ing
he moplas ic p ocessing a e unclea . Thus, he eac i i y o p o eins depends
on hei physicochemical en i onmen as well as on he he momechanical
ea men used (He nandez-Izquie do and K och a 2008).
Sol en cas ing
Fo he o ma ion o p o ein ilms o coa ings, he p o ein has o be i s
dissol ed in a p ope sol en , which some imes equi es hea ing o pH
Backg ound. P oduc s om p o eins
137
adjus men , as well as he addi ion o some compounds which could imp o e
ilm- o ming o o he p ope ies
Subsequen ly, he mix u e is hea ed abo e he lipid mel ing poin and
hen homogenized. Degassing is an impo an s ep o elimina e bubble
o ma ion in he inal ilm o coa ing. Finally, he p o ein ilm o coa ing is
o med by applying he p epa ed o mula ion o he desi ed cas ing o p oduc
su ace and allowing he sol en o e apo a e. P o iding hea ed ai a low
humidi y and high eloci y inc eases d ying a es (K och a 2002).
The mo-mechanical p ocessing
Comp ession moulding
Comp ession moulding echnique use he combina ion o high
empe a u es, high p essu es, sho imes, and low mois u e con en s in o de
o he ans o m he p o ein-plas icize blends in o iscoelas ic mel s. Then,
he p o ein-based bioplas ics a e o med by cooling, inc easing hyd ogen,
ionic, hyd ophobic, and co alen in e ac ions (He nandez-Izquie do and
K och a 2008). The use o highe comp ession moulding empe a u es
ypically p omo es a mo e ex ensi e p o ein dena u a ion, and as a
consequence highe c oss-linking.
Comp ession moulding can esul in he o ma ion o p o ein-based ilms
o ma e ials whose mechanical and ba ie p ope ies a e dependen on he
o mula ion and p ocessing condi ions used. This echnology is sui able o
in es iga ing he he moplas ic p ope ies o plas icized p o eins as well as he
p ope ies o he esul ing ilms and ma e ials (He nandez-Izquie do and
K och a 2008).
Backg ound. P oduc s om p o eins
144
necking/yielding bu ei he emains app oxima ely cons an o ises less
s eeply wi h inc easing s ain, depending on he ex en o cold d awing.
Cold d awing succeeds he yield poin whe e ma e ial unde goes
pe manen de o ma ion as a esul o molecula slippage. Con inuing
ex ension o he na ow po ion o he dumbbell specimen is achie ed du ing
d awing by causing he shoulde s o he neck o a el along he specimen as
i educes om he ini ial c oss-sec ion o he d awn c oss-sec ion. A u he
elonga ions, he slope o he s ess-s ain cu e inc eases again, due o “s ain
ha dening”/“molecula o ien a ion”, and inally ma e ial ailu es (Akay 2012).
This ype o cu e is e y use ul since allows o classi y he ma e ial. Fo
ins ance, Figu e 3.4-4 compa es ou di e en plas ic ma e ials:
Figu e 3.4-4: compa ison be ween di e en cu es s ess-s ain
Thus we can say ha (a) is a low duc ili y polyme , (b) is a duc ile polyme ,
(c) is a duc ile polyme capable o cold d awing, and (d) is a polyme wi h long-
ange elas ici y (Akay 2012).
Applica ions o p o ein bioplas ics
The main goal o biodeg adable bioplas ics is o achie e he eplacemen
o exis ing syn he ic, non-biodeg adable p oduc s o hese o he s a he

Backg ound. P oduc s om p o eins
145
lowes cos possible. O he goals may be also ound o speci ic applica ions.
Fo ins ance, edible ilms and coa ings aim o imp o ing ood quali y and shel
li e by educing he e ec o mois u e, oxygen, mig a ion, e c., p o ec ing ood
om mic obes, main aining ood p oduc in eg i y, and enhancing p oduc
appea ance. Howe e , his mus be ela ed o he cos o coa ing ma e ials
and o he cos o he coa ing p ocess.
Ce ain new edible ilm and coa ing ma e ials made om p o eins a e
a ge ed o eplacing ma e ials cu en ly used in exis ing applica ions.
Howe e , compa ed o he la ge numbe o s udies pe o med on ilm
o ma ion and p ope ies, a ela i ely small numbe o applica ion s udies
ha e been pe o med. Fo his eason, in o ma ion a ailable is gene ally
lacking on app oaches o coa ing oods, as well as he esul ing e ec i eness
o edible ilms and coa ings in ood sys ems. This makes i e y di icul o
ood p ocesso s o decide on he “ alue-added” me i o an edible ilm o
coa ing ela i e o he addi ional cos in ol ed (K och a 2002).
Composi e ma e ials
A composi e compound is a ma e ial made by combining wo o mo e
ma e ials, and equen ly hey ha e e y di e en p ope ies. The wo
ma e ials wo k oge he o gi e he composi e unique p ope ies. Howe e ,
wi hin he composi e you can easily ell he di e en ma e ials apa as hey
do no dissol e o blend in o each o he .
Na u al composi es exis in bo h animals and plan s om he ea ly
beginnings. Thus, wood is a composi e which is made om long cellulose ib es
(a polyme ) held oge he by a much weake subs ance called lignin. The wo
weak subs ances (lignin and cellulose) oge he o m a much s onge
s uc u e.
Backg ound. P oduc s om p o eins
146
Nowadays, human ha e syn he ized his kind o compounds. The i s
mode n composi e ma e ial was ib eglass. I is s ill widely used oday o boa
hulls, spo s equipmen , building panels and many ca bodies. The ma ix is a
plas ic and he ein o cemen is glass ha has been made in o ine h ead. On
i s own, he glass is e y s ong bu b i le and i will b eak i ben sha ply. The
plas ic ma ix holds he glass ib es oge he and also p o ec s hem om
damage by sha ing ou he o ces ac ing on hem.
Some ad anced composi es a e now made using ca bon ib es ins ead o
glass. These ma e ials a e ligh e and s onge han ib eglass bu mo e
expensi e o p oduce. They a e used in ai c a s uc u es and expensi e
spo s equipmen . Ca bon nano ubes ha e also been used success ully o
make new composi es.
147
A Albe o y An onio
Po ayuda me a c ece jun o a ellos,
Po se una pieza cla e en mi o mación como in es igado , po odas esas
ho as de abajo que hemos pasado jun os.
149
4. Ma e ials and Me hods

Ma e ials and Me hods. Ma e ials
151
4.1. Ma e ials
C ay ish powde
The CF mea was sepa a ed om he shell by g inding and sie ing and
supplied as CF pulp by ALFOCAN (Isla Mayo , Se illa, Spain). Figu e 4.1-1 shows
he p ocess ollowed o ob ain di e en p o ein ac ions om c ay ish (CF)
mea . CF pulp was kep ozen un il i s use. A e hawing a 4⁰C, CF pulp was
homogenized and subjec ed o cen i uga ion a 15,000 x g o 15 minu es in
a Cen omix II-BL (Selec a, Spain), ob aining h ee di e en phases: a hea y
phase, CF1P (c.a. 20 w . %), an in e media e phase (CF2P) which is he aqueous
phase (c.a. 70 w . %) and a ligh phase, CF3P (c.a. 10 w . %). The CF2P was he
selec ed phase because i is he wa e soluble p o ein ac ion and i
ep esen s he highes p o ein con en . Finally, he in e media e phase (CF2)
was eeze-d ied in a BETA 1-8 LD Plus Se ies (CHRIST, Ge many) o ob ain a
powde - ac ion ich in p o eins. This phase will be named CF2L.
Figu e 4.1-1 : Diag am o he p ocedu e ca ied ou in o de o ob ain he CF2L
p o ein concen a e.
Ma e ials and Me hods. Ma e ials
152
The p o ein con en o he CF2L was de e mined in quad uplica e as % N
x 6.25 using a LECO CHNS-932 ni ogen mic o analyse (Leco Co po a ion, S .
Joseph, MI, USA) om Mic oanalysis se ice (CITIUS, Uni e si y o Se ille). In
he same way, lipid, mois u e and ash con en s we e de e mined acco ding o
A.O.A.C. (2000).Table 4.1-1 shows he elemen al cha ac e isa ion o CF2L
sys em:
Componen
w . %
P o ein
Mois u e
Lipid
Ash
78.6 ± 0.5
6.8 ± 0.1
5.1 ± 0.3
9.5 ± 0.6
Table 4.1-1: Elemen al cha ac e isa ion o CF2L sys em
Fi s o all, i is ema kable he high p o ein pe cen age (ca. 80 w . %).
Hence, acco ding o Pea son classi ica ion (1983), i mus be conside ed as a
p o ein concen a e. Fu he mo e, his sys em con en s up o 5 w . % o lipids
despi e cen i uga ion s age was ca ied ou . Tha means ha his s age is no
able o a comple e o ganic and aqueous phases sepa a ion, o in he manual
p ocedu e o sepa a ing bo h phases a ce ain amoun o o ganic phase was
adjoined oge he o aqueous phase.
All sys ems s udied ha e in common his p o ein concen a e, howe e
o each p oduc (emulsion, gel o bioplas ic) some modi ica ions ha e been
conside ed: o emulsions a polysaccha ide (a gum) ha e been used o p o ide
s abili y. In addi ion, o gels h ee hyd olysed sys ems ha e been ob ained a
h ee di e en deg ee o hyd olysis and o bioplas ics wi h a low- alue
c ay ish lou ha e been e alua ed in o de o be cos -compe i i e wi h
syn he ic polyme s.
Ma e ials and Me hods. Ma e ials
153
Ma e ials o Emulsions
The oil-in-wa e emulsions consis o a con inuous phase o dis illa e
wa e and dispe sed phase o high oleic oil. To p o ide s abili y, apa om
he ini ial c ay ish p o ein concen a e (CF2L), a polysaccha ide (xan han gum)
was used in o de o imp o e he sys em s abili y. Gums ha e a high in luence
on he s uc u al cha ac e is ics o ood p oduc s, modi ying he ex u e and
he o ganolep ic p ope ies, e en being p esen a concen a ions lowe han
1 w . %. Fo his eason, gums a e nowadays widely used o a eplacemen
in many low-calo ie p oduc s (Williams and Phillips 2003). Xan han gum was
disco e ed in he 1950s and nowadays ha e widely applica ion in he ood
indus y, whe e was in oduced in he ea ly 1970s. This gum is ob ained om
he genus Xan homonas by ae obic e men a ion
Figu e 4.1-2 shows he p ima y s uc u e o he Xan han gum (XG):
Figu e 4.1-2: P ima y s uc u e o xan han gum
. The xan han gum is an anionic molecule which has a (1,4)-β-D-
glucopy anose backbone and a isaccha ide side chain on e e y o he glucose
esidue linked h ough he C3 posi ion. The side chain consis s o wo
mannopy anosyl esidues linked on ei he side o a glucu opy anosyl u onic
acid g oup. The inne mannose esidue which is connec ed o he backbone
may be ace yla ed while he e minal mannose esidue may be py u a ed. The
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256
con i m ha DPPH measu emen s a e no dependen on pH o on he
hyd olysis deg ee.
Finally, a pH 8.0 he lowes an ioxidan ac i i y was ound o FC eagen .
The deg ee o hyd olysis seems o a ou FC ac i i y in some ex en a his pH,
excep o i s highes alue (i.e. CF2LH120)
I is wo h poin ing ou ha he di e en beha iou o p o ein sys ems
could be s iking because i depends on he me hod used. In ac , many s udies
ha e been epo ed ha he an ioxidan ac i i y depends on he na u e o he
e e ence compound (DDPH, ABTS, FC) (Wojdylo, Oszmianski e al. 2007,
Woo on-Bea d, Mo an e al. 2011, Boulanoua , Abdelaziz e al. 2013). In any
case, ega dless o he pH s udied and he di e en e e ence compounds
used, he e seems o be a gene al endency o inc easing an ioxidan ac i i y
wi h he deg ee o hyd olysis, e en hough is no always signi ican ly de ec ed.
An excessi ely high deg ee o hyd olysis seems o lose his abili y.
Figu e 5.3-20 shows TCA-soluble pep ides ( ee-amino acids and sho
pep ides) be o e and a e he mal p ocessing. The p o eoly ic ac i i y o
enzymes, which a e capable o b eaking he chemical bonds o he muscle
ib es du ing he cooking s ep, has been widely s udied. This enzyma ic
p ocess may be impo an since i can modi y he cha ac e is ic ex u e o
esh ish o ood p oduc s (Mille and Spinelli 1982, G eene and Babbi 1990)
The de e mina ion o TCA-soluble pep ides has been ca ied ou by
means o measu ing he abso bance o TCA-soluble pep ide solu ion be o e
and a e gela ion p ocess a 280 nm. Fi s o all, i can be obse ed signi ican
di e ences be ween non-hyd olysa e sys em (CF2L) and hyd olysa es sys ems
(CF2LH5, CF2LH25 and CF2LH120). These esul s could be expec ed since hey
can be a consequence o he hyd olysis ca ied ou . This means ha an

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257
inc ease in pH alues gi es aise an inc ease in he numbe o amino acids and
small pep ides in he bulk solu ion.
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
AG
2.0 6.5
CF2L CF2LH5 CF2LH25 CF2LH120
CF2L CF2LH5 CF2LH25 CF2LH120
pH 8.0
BG
Abso bance
Figu e 5.3-20: P o eoly ic ac i i y measu ed a 280 nm in all he sys ems s udied
(CF2L, CF2L5, CF2L25 and CF2L120), e alua ed a h ee di e en pH alues (2.0, 6.5 and
8.0), be o e and a e gela ion p ocess.
Fo CF2L sys em, a sligh inc ease o TCA-soluble pep ides a 20⁰C can be
obse ed when an inc ease o pH akes place. Finally, a e gela ion p ocess,
TCA-soluble pep ides do no inc ease in any case, o his eason we can
disca d p o eoly ic ac i i y as a consequence o he he mal p ocedu e
ollowed.
Rega ding o hyd olysa es sys ems, we could no ound any pH
dependence o TCA-soluble p o eins on pH o hyd olysa es sys ems, nei he
be o e gela ion no a e gela ion. Thus, p o eoly ic ac i i y was no ound in
any sys em, and i is no pH-dependen .
Finally, i is ema kable he dec ease o TCA-soluble pep ides ound a pH
6.5 a e gela ion p ocess o CF2L sys em. Hence, a his pH, he gel no only
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258
exhibi be e mechanical p ope ies (highe gel s eng h and high WHC), bu
also he gel is able o hold in a g ea e ex end ee amino acids and sho
pep ides in i s s uc u e. This can be ela ed o he highes alues o WHC
ound o CF2L sys em a pH 6.5.
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259
5.4. C ay ish-based bioplas ics
An indus ial low cos su plus ob ained om c ay ish (CF) (d ied in a
o a y u nace) was used in his sec ion due o he es ic ed a ailabili y o
CF2L p o ein concen a e, along wi h he high aw ma e ial equi ed o
p o ein-based bioplas ics manu ac u ing. This way o p oceeding allows us o
s udy he op imisa ion o CF-based bioplas ic.
E en ually, a he end o his sec ion, esul s we e compa ed wi h CF2L-
bioplas ics p ocessed by using he p o ocol ob ained om CF-based
bioplas ics.
The i s s age o all p o ein-based bioplas ics is o selec he a ion
p o ein/plas icize . This elec ion is usually made based on he p ocessabili y
o he dough-like ma e ial ob ained.
Figu e 5.4-1 shows bo h o que and empe a u e p o iles as a unc ion o
mixing ime o blends ob ained a di e en CF/GL a ios, as well as hei isual
appea ance a e mixing.
These esul s pu o wa d he ema kable dependence o hese
pa ame e s on he CF/GL a io. Thus, a apid inc ease in o que up o a
maximum alue akes place o he sys em ha ing less amoun o plas icize ,
deno ed as 80/20, which ep esen s he CF/GL weigh a io. This e olu ion is
eadily ollowed by an asymp o ic dec ease owa ds a pla eau alue. In
con as , he sys em 70/30 shows a mode a e g ow h in o que showing no
maximum alue bu a slow endency o he pla eau alue. The p o ile o he
60/40 sys em shows only a sligh ly ini ial inc ease in o que, being domina ed
by a cons an o que alue o e mixing ime. In gene al, he empe a u e
e olu ion is e y simila o he o que p o ile whe e an inc ease akes place
excep ing o he lowes CF/GL a io. Bo h inc eases in o que and
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260
empe a u e may be a ibu ed o shea -induced exo he mic eac ions
de eloped du ing he mixing p ocess.
020 40 60
0
10
20
30
40
50
To que (N·m)
Time (min)
20
30
40
50
60
70
80
90
CF/GL Temp.(ºC) To que (N m) SME(kJ kg-1)
(60/40) 47901
(70/30) 33861
(80/20) 1331
Tempe a u e (ºC)
Figu e 5.4-1: Images, SME and e olu ion wi h ime o mixing o que and empe a u e
o sys ems CF/GL (80/20, 70/30 and 60/40).
As a consequence o he abo e-men ioned di e ences in o que p o ile,
he speci ic mechanical ene gy (SME) employed o mixing is also qui e
di e en . This cha ac e is ic pa ame e o mixing may be de ined as ollows
EQ. (5.4-1). (Redl, Mo el e al. 1999):
𝑆𝑀𝐸=𝜔
𝑚∫ 𝑀(𝑡)𝑑𝑡
𝑡𝑚𝑖𝑥
0
(5.4-1)
whe e ω (in ad/s) is he mixing speed, m (in g) he sample mass, M( ) (in
Nm) he o que and mix (in s) he mixing ime. The alues o he SME o hese
h ee sys ems a e included in Figu e 5.4-1. A ema kable inc ease in his
pa ame e can be obse ed wi h he CF/GL a io. This e ec akes also place
a he highe concen a ions in spi e o he ac ha he pla eau o que alues
a e ela i ely close.
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261
F om he isual appea ance obse ed in Figu e 5.4-1, he 80/20 sys em
does no seem o con ain enough amoun o plas icize o ob ain an easy- o-
handle ma e ial, gi ing ise o a g anula ed powde ins ead o a homogeneous
dough-like blend. This is e idenced by he ac ha his is he sys em displaying
he highes o que alues, which co espond o he highes heological
consis ency. On he o he hand, he highes amoun o glyce ol in he 60/40
sys em p o ides a nea dough-like appea ance o he blend. This is also
consis en wi h he lowes o que alues ob ained (associa ed o a lowe
consis ency) a e he mixing p ocess. The 70/30 blend shows in e media e
appea ance be ween bo h limi s al hough i is close o he mos concen a ed
p o ein-based bled. In he p esen s udy, he sys em 80/20 has been disca ded
because he high ene gy equi ed o mixing. Mo eo e , his sys em has
p o en o be ha d o injec ( esul s no shown). On he o he hand, sys ems
con aining a low p o ein/glyce ol a io may be injec ed easily bu he inal
bioplas ic ma e ials end o exude he excess o glyce ol. Exuda ion is a well-
known phenomenon desc ibed in he polyme /plas icise li e a u e and
should be a oided in o de o p e en con amina ion o he su ounding
ma e ials (e.g. in ood packageing) (Rahman and B azel 2004). This seems o
be he case o he 60/40 sys em. As a esul , he 70/30 sys em is selec ed as
he mos sui able o ob ain a homogeneous and easily injec able CF/GL blend.
In o de o compa e and e alua e he e ec o chemical agen s and
syn he ic polyme on c ay ish-based bioplas ic ma e ials, a dual s a egy was
ollowed. On he one hand he p esence o sodium sulphi e (SS) o bisulphi e
(BS) as educing agen s, u ea (U) as dena u ing agen and L-cys eine (LC) as
c osslinking agen we e analysed as chemical modi ie s. On he o he hand, a
polyes e om ossil sou ce, which has been widely used as he polyme
ma ix in he de elopmen o new ma e ials, which is highly lexible,

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biodeg adable, biocompa ible and easy o p ocess, was used
(polycap olac one, PCL).
C ay ish-bioplas ic wi h chemical
modi ie s
Blends cha ac e isa ion
P epa a ion o blends by he moplas ic mixing
Figu e 5.4-2 shows he e olu ion o o que as a unc ion o ime o
di e en 70/30 CF/GL blends p epa ed using SS as educing agen , U as
dena u ing agen , o LC as c osslinking p omo e a di e en concen a ions
(3 and 30 mg/g p o ein o SS and U and 1 and 3 mg/g o LC).
0 5 10 15 20 25
0
5
10
15
20
25
30
AMOUNT OF ADDITIVES
Sys em 0 1g kg-1 3g kg-1 30g kg-1
CF/GL - - -
CF/GL SS - -
CF/GL U - -
CF/GL LC - -
To que (N m)
Time (min)
Figu e 5.4-2: E olu ion wi h ime o mixing o que o addi i e-con aining sys em (SS,
U and LC).
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263
These esul s gene ally show ha any o he addi i es used induces an
an icipa ion o he o que p o ile, pa icula ly a he highes addi i e
concen a ions. No di e ences in he o que p o ile a e no iced a he wo
di e en concen a ions o BS (da a no shown). The highes amoun o
addi i e (co esponding o SS o U) also yields lowe o que pla eau alues,
which may be conside ed bene icial o he p ocessabili y o blends, o
example unde injec ion moulding. Tempe a u e- ime p o iles show he same
ype o e olu ion o all he addi i es and concen a ions in spi e o ha an ai
s eam was used as a cooling luid. No di e ences a e ound in he inal alues
o he highes SS o U con en in his case. The only di e ence ound be ween
empe a u e and o que p o iles is ha he o me unde go a u he delay in
ime, which may be ela ed o he ai -cooling e ec .
I may be also no ed ha he sys em is no s ill p ope ly mixed a imes
sho e han ha one co esponding o he maximum o que. The e o e, as a
gene al ule, he mixing ime ange o subsequen blend p ocessing is
selec ed once he pla eau o que alue is eached such ha a good
homogenei y deg ee is assu ed.
Ano he in e es ing pa ame e o compa e di e en mixing p ocesses is
he ela i e ene gy inpu (REI), which is de ined as ollows (5.4-2).
𝑅𝐸𝐼 (%)=𝑆𝑀𝐸𝑚𝑖𝑛(𝑎,𝑐)
𝑆𝑀𝐸𝑚𝑖𝑛(0)·100
(5.4-2)
whe e 𝑆𝑀𝐸𝑚𝑖𝑛(0) and 𝑆𝑀𝐸𝑚𝑖𝑛(𝑎,𝑐) a e he SME alues a which o que
eaches 95% o he maximum alue o he p o ile o he addi i e ee blend
and addi i e a concen a ion c, espec i ely. The e o e, REI is an index o he
educ ion in ene gy inpu ela i e o ha one equi ed o mixing he addi i e
ee blend.
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264
The alues o SME20 and REI pa ame e s a e shown in Figu e 3.1-1 o
hese addi i es. The SME alues o BS a e also included in his igu e SME20 is
he speci ic mechanical ene gy supplied, as de ined in EQ. (5.4-2), a e mixing
o 20 min. As may be obse ed in his g aph, an inc ease in addi i e
concen a ion always leads o an appa en inc ease in SME20 pa ame e , as a
consequence o he abo e-men ioned an icipa ion o he o que p o ile. I he
same addi i e concen a ion is used, his inc ease is pa icula ly ema kable
o LC and, in any case, is only mode a e when BS is used.
0
5000
10000
CF/GL 1g kg-1 3g kg-1 30g kg-1
(70/30)
SME20 (kJ kg-1)
Adi i e
SS
BS ULC
Figu e 5.4-3: Speci ic mechanical ene gy (SME) employed o mixing a 20 minu es
The alues o REI pa ame e s a e shown in Figu e 5.4-4 o hese
addi i es.
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265
Adi i e
SS
BS ULC
0
50
100
150
CF/GL 1g kg-1 3g kg-1 30g kg-1
(70/30)
REI (%)
Figu e 5.4-4: Reduc ion ene gy index (REI) o each sys em.
The esul s ob ained wi h pa ame e REI con i m he ele an e ec o
he addi ion o LC a cons an addi i e concen a ion. Thus, es ima ion
endencies om he esul s ob ained indica e ha he addi i e/p o ein a io
equi ed o achie e a 50% REI would be 33 mg/g o U and 27 mg/g o SS,
whe eas a a io as low as 4.4 mg/g would be equi ed by using LC.
Rheological cha ac e isa ion o blends
Figu e 5.4-5 shows he dependence o linea iscoelas ic unc ions on
empe a u e o di e en blends con aining 3 mg addi i e pe g CF p o ein.
The addi i e ee (CF/GL) blend is also displayed in his igu e.
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272
Uniaxial ensile s eng h measu emen s
Figu e 5.4-9 displays he esul s o s ess-s ain cu es ob ained om
ensile s eng h measu emen s o addi i e- ee and addi i e-con aining
specimens a an addi i e/CF a io.
0.00 0.01 0.02
0.0
0.2
0.4
0.6
0.8 Sys em
CF/GL
CF/GL BS
CF/GL SS
CF/GL U
CF/GL LC
 (MPa)
(mm/mm)
Figu e 5.4-9: S ess e sus s ain cu es om ensile s eng h measu emen s o
di e en CF/GL and addi i e-con aining p obes.
All he cu es exhibi a simila beha iou which consis o an ini ial linea
elas ic beha iou o high cons an s ess-s ain slope yielding high alues o
he Young’s modulus (E), ollowed by a de o ma ion s age wi h a con inuous
dec ease in he s ess-s ain slope. A second cons an slope is eached a he
end o he plas ic de o ma ion s age. All he cu es e en ually each a
maximum alue o he s ess (σmax) and he elonga ion a b eak (εmax). Only

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273
LC leads o an appa en enhancemen o he ensile s eng h p o ile, also
leading o a sligh ly sho e s ain alue.
The alues o s ess-s ain pa ame e s (E, σmax and εmax) and hei
co esponding s anda d de ia ions a e plo ed in Figu e 5.4-10 o he
addi i e- ee and addi i e-con aining specimens a 3 mg/g addi i e/CF a io.
0.5
1.0
1.5
2.0
2.5 max
max
E
max (%)
max (MPa)
,
0
20
40
60
80
100
LC
130ºC
LC
600s
LC
U
SS
BS
E (MPa)
CF/GL
Figu e 5.4-10: Pa ame e s om ensile s eng h measu emen s: Maximum s ess
(σmax), elonga ion a b eak (εmax) and Young’s’ modulus (E) o di e en CF/GL and
addi i e-con aining p obes.
This igu e pu s o wa d once again ha addi i e LC is he only one ha
imp o es pa ame e s E and σmax o e he addi i e- ee sys em, unde he
same p ocessing condi ions. On he o he hand LC-added specimens exhibi
lowe alues o εmax. The es o addi i es lead o simila o e en lowe alues
o he h ee pa ame e s. Figu e 5.4-10 also shows he alues o LC-added
specimens moulded a longe ime o highe empe a u e. As may be
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274
obse ed, an inc ease in he packing ime om 200 s up o 600 s does no yield
any no iceable change in ensile pa ame e s. This esul indica es ha a e
200 s packing ime, he p obe is al eady closed by he solidi ied blend. On he
o he hand, an inc ease in he mould empe a u e leads o ema kable
changes in ensile pa ame e s. Thus, he maximum s ess and abo e all he
elonga ion a b eak unde go an appa en inc ease in alue (ca. 12 and 70%,
espec i ely), whe eas he Young’s modulus clea ly dec eases (ca. 30%) by
inc easing he mould empe a u e om 100 o 130 0C. In e es ingly, i is he
elonga ion a b eak he p ope y ha unde goes he mos ema kable
enhancemen by a ou ing hea -induced c osslinking. In his way, he ma e ial
exhibi s highe oughness, in spi e o being less s ong. In ac , as s a ed by
Lag ain e al. (2010), inc easing he elonga ion a b eak o glassy, amo phous
polyme s ypically goes a he expense o he elas ic modulus. This
compensa ion may also a ec o he bending elas ic p ope ies o he
bioplas ic, hus explaining he small dependence o DMA p o iles on moulding
empe a u e.
Mo eo e , his beha iou is simila o ha one ound o o he
elas ome ic ma e ials such as ubbe -based blends (Za a e-Rami ez, Ma inez
e al. 2011) and is consis en wi h he esul s om DMA measu emen s ha
show an ex ension o he ubbe y pla eau ob ained a high empe a u e.
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275
C ay ish-bioplas ic wi h a syn he ic
polyme . Composi e ma e ials
A syn he ic biodeg adable polyme (PCL) we e used o imp o e
mechanical p ope ies o CF-based bioplas ic. This s udy can be conside ed as
an al e na i e o he use o addi i es (p e ious sec ion).
Blends cha ac e isa ion
P epa a ion o blends by he moplas ic mixing
Figu e 5.4-11 exhibi s o que and empe a u e p o iles as a unc ion o
mixing ime o di e en CF/GL/PCL blends main aining he same CF/GL a io
a ca. 2.3, using di e en PCL con en , whe e he blend wi hou PCL is used as
he e e ence sys em.
020 40 60
0
5
10
15
20
25
30
35
40
45
50
CF/GL/PCL To que(Nm) Temp. (ºC)
(70/30/0)
(63/27/10)
(49/21/30)
Time (min.)
To que (Nm)
20
30
40
50
60
70
80
90
100
110
Tempe a u e (ºC)
Figu e 5.4-11: E olu ion o o que and empe a u e o e he mixing p ocess o
c ay ish lou /glyce ol/polycap olac one (CF/GL/PCL) sys ems: a cons an CF/GL
a io: (70/30/0), (63/27/10) and (49/21/30)
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276
These esul s pu o wa d he ele an dependence o o que and
empe a u e on he CF/GL/PCL a io. Thus, a apid inc ease in o que up o a
maximum alue akes place, ollowed by an asymp o ic dec ease owa ds a
pla eau alue. Tempe a u e p o iles gene ally ollow an inc ease owa ds a
pla eau alue. The ime equi ed o each he pla eau alues o bo h a iables
( o que and empe a u e), which is oughly he same, is clea ly dependen on
he CF/GL/PCL a io. This coincidence may be seen as a consequence o he
de elopmen o exo he mic c osslinking eac ions du ing he mixing p ocesses
ha in ol e bo h an inc ease in empe a u e and consis ency ( e lec ed in
o que). Bo h p o iles show also an ini ial induc ion pe iod, being mo e
e iden o hose blends displaying he slowes e olu ion. I is wo h
men ioning ha a o que peak appea s when unmel ed PCL is p esen in he
blend. Thus, he PCL mel poin , which is eached a abou 55 0C, is coinciden
wi h he maximum o que alue. In o he wo ds, he o que does no s a o
dec ease un il he PCL mel ing poin is exceeded.
As may be also obse ed in his igu e, an inc ease in PCL con en leads
o a as e o que and empe a u e kine ics and, as a consequence, o an
an icipa ion o bo h p o iles. The e ec also gi es ise o a gene al inc ease in
o que and empe a u e alues a any ime.
Figu e 5.4-12 exhibi s o que and empe a u e p o iles as a unc ion o
mixing ime o di e en CF/GL/PCL blends. This igu e shows bo h p o iles o
blends con aining 10% PCL a di e en CF/GL a ios.
As may be obse e in his igu e, he beha iou is qui e simila o he
p e iously ound o sys ems a cons an CF/GL a io. Again, he empe a u e
eached is abo e he PCL mel ing poin , which ensu es a homogenous mixing.
Howe e , he beha iou ound o he sys em (60/30/10), seems o ha e an
excess o glyce ol, which lead o delay he pla eau alue.
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277
020 40 60
0
5
10
15
20
25
30
35
40
45
50
CF/GL/PCL To que(Nm) Temp. (ºC)
(65/25/10)
(63/27/10)
(60/30/10)
Time (min)
To que (Nm)
20
30
40
50
60
70
80
90
100
110
Tempe a u e (ºC)
Figu e 5.4-12: E olu ion o o que and empe a u e o e he mixing p ocess o
c ay ish lou /glyce ol/polycap olac one (CF/GL/PCL) sys ems: a cons an PCL
concen a ion: (63/27/10), (60/30/10) and (65/25/10).
The mal cha ac e iza ion o blends
Hea low pa e ns ob ained om Di e en ial Scanning Calo ime y (DSC)
measu emen s a e shown in Figu e 5.4-13 and Figu e 5.4-14. Figu e 5.4-13
shows he he mog am o CF lou and o he e e ence sys em (CF/GL/PCL,
70/30/0), as well as he p o iles co esponding o CF/GL/PCL blends a
cons an CF/GL a io, as a unc ion o PCL con en . On he o he and, Figu e
5.4-14 displays he DSC esul s o CF/GL/PCL blends con aining 10% PCL as a
unc ion o he CF/GL a io.

Resul s
278
25 50 75 100 125
Sys em Symbol
CF Flou
CF/GL (70/30)
CF/GL/PCL (63/27/10)
CF/GL/PCL (49/21/30)
Exo Up
Hea Flow (W/g)
Tempe a u e (ºC)
Figu e 5.4-13: DSC p o iles o c ay ish lou and sys ems a cons an CF/GL a io:
(70/30/0), (63/27/10) and (49/21/30) (A)
25 50 75 100 125 150
Sys em Symbol
CF/GL/PCL (65/25/10)
CF/GL/PCL (63/27/10)
CF/GL/PCL (60/30/10)
Hea Flow (W/g)
Tempe a u e (ºC)
Exo Up
Figu e 5.4-14: DSC p o iles o sys ems a cons an PCL concen a ion: (63/27/10),
(60/30/10) and (65/25/10).
Resul s
279
CF lou displays a ypical endo he m o a ai ly dena u ed p o ein sys em.
This p o ile exhibi s an endo he mic i s peak a 68 0C, a glass ansi ion (Tg)
a ca. 92 0C, as well as a b oad endo he mic e en be ween he Tg and 130 0C.
The i s he mal e en can be a ibu ed o he physical ageing e ec , which
was p e iously epo ed o his CF lou (Fa ahnaky, Gue e o e al. 2008).
Physical ageing is a gene al phenomenon ha occu s o e ime in glassy o
pa ial glassy polyme s below hei Tg and is a mani es a ion o he non-
equilib ium na u e o he glassy s a e (S ink 1978, Anon 1997). The glass
ansi ion a a ound 90 0C is consis en wi h p e ious esul s epo ed by
Fa ahnaky e al. (2008) o c ay ish lou and Hashimo o e al. (2004) o ish
muscle p o eins. On he o he hand, he b oad endo he mic e en may be
ela ed o he huge a ie y o p o ein ac ions o di e en molecula weigh
ha cons i u e he CF lou a epo ed in a p e ious pape (Rome o, Co dobes
e al. 2011).
Wi h ega d o he endo he ms ob ained o he e e ence sys em he
wo endo he mic e en s anish. The o al disappea ance o he i s
endo he mic peak, mos p obably as a consequence o mixing, con i ms i s
physical ageing-d i en na u e. Howe e , he glass ansi ion emains oughly
a he same empe a u e o all he blends s udied.
In addi ion, i is wo h men ioning ha all he sys ems con aining PCL
display an appa en endo he mic peak a ca. 55 0C, which is a ibu ed o he
PCL mel ing poin . This peak becomes mo e p onounced wi h inc easing PCL
con en .
The esul s ob ained om DSC measu emen s also con i m he sui abili y
o he empe a u e selec ed o he cylinde and mould, since he o me (60
0C) is highe han he endo he mic peak co esponding o he PCL mel ing
whe eas he la e empe a u e (100 0C) is highe han he glass ansi ion in
Resul s
280
o de o a ou mobili y and empe a u e-induced p o ein c osslinking (in
combina ion wi h p essu e).
Injec ion moulding p ocess
Table 5.4-2 shows he condi ions selec ed o he injec ion moulding
p ocess o each o he blends s udied.
T (0C)
P essu e (MPa)
Time (s)
P e-injec ion cylinde
Injec ion
Packing s age
60
60
100
100
0.1
0.1-50
50
20
100
<1
20
200
Table 5.4-2: Injec ion moulding pa ame e s.
The p ocessing pa ame e s ( empe a u e, p essu e and ime) alues o
he injec ion moulding p ocess used in his s udy, which a e simila o hose
used in he p e ious sec ion. The alues o he p ocessing pa ame e s a he
p e-injec ion cylinde a e selec ed o ensu e a blend iscosi y low enough o
acili a e i s injec ion in o he mould. The esidence ime selec ed o he
packing s age ( igh a e injec ion) has been 220 s since no u he
enhancemen has been no iced by inc easing his pe iod. In addi ion,
exposi ion o high empe a u es o a long ime ypically leads o p o ein
deg ada ion (i.e. ia Mailla d- ype eac ions) (Fayle, Ge a d e al. 2002).
Biocomposi e cha ac e isa ion
Dynamic Mechanical Tempe a u e Analysis
Figu e 3.1-1 shows he alues o he elas ic modulus, E’ om DMA
empe a u e amp es measu emen s. The e e ence sys em is compa ed
Resul s
281
wi h hose specimens wi h he same p o ein/plas icise a io (CF/GL/PCL:
63/27/10 and 49/21/30).
As may be obse ed, all he specimens s udied show a simila p o ile o
E’ a low and medium empe a u e. In his way, an inc ease in empe a u e
leads o a dec ease in elas ic modulus (E’) ha ends o each a pla eau alue.
Howe e , i is ema kable ha PCL con aining samples exhibi highe elas ic
modulus p o ided ha empe a u e emains below he PCL mel ing poin . A
he mel ing poin he h ee samples show a he coinciden alues ha
unde go a dec ease wi h inc easing empe a u e. In ac , he e is a egion
whe e he PCL- ee specimen shows sligh ly highe E’ alues. E en ually a
empe a u e is eached abo e which he dec ease in E’ ound o he e e ence
specimen becomes as e whe eas he PCL-con aining composi es end o
each a pla eau egion.
-20 0 20 40 60 80 100 120 140
104
105
106
107
108
109
1010
Sys em E' (Pa)
CF/GL/PCL (70/30/0)
CF/GL/PCL (63/27/10)
CF/GL/PCL (49/21/30)
E' / (Pa)
Tempe a u e (ºC)
Figu e 5.4-15: S o age modulus om DMA empe a u e amp measu emen s
pe o med a cons an equency (6.28 ad/s) and hea ing a e (3 0C/min) o
CF/GL/PCL p obes a cons an CF/GL a io: 70/30/0, 63/27/10, 49/21/30.
Resul s
288
0
5
10
200
400
600
800
(49/21/30)
(63/27/10)
(55/15/30)
(65/25/10)
(60/30/10)
(40/30/30)
2.6
2.3
2
0% PCL
10% PCL
30% PCL
Toughness (kJ/m3)
1.3
Ra io CF/GL
(70/30/0)
Figu e 5.4-22: Toughness om s ess-s ain cu e o all CF/GL/PCL sys ems:
(70/30/0), (65/25/10), (63/27/10), (60/30/10), (55/15/30), (49/21/30) and
(40/30/30).
As may be obse ed, highe alues in E’ and especially in εmax and
oughness a e ob ained by inc easing PCL om 0 o 30%.
Mo eo e , an inc ease in he CF/GL a io yields a di e en beha iou
depending on PCL con en . Those specimens con aining 10 w . % PCL display
a maximum alue in E as well as a minimum alue in εmax. As a esul , a
mode a e inc ease in oughness akes place. I should be also aken in o
accoun ha an inc ease in he CF/GL a io also in ol es a dec ease in he
PCL/CF a io. These esul s sugges ha bo h a ios exe opposi e e ec s,
whe e he dominan e ec seems o be he o me a low CF con en and he
la e a high CF con en . This balance can explain he occu ence o he
maximum in E and minimum in εmax.
On he o he hand, an inc ease in PCL con en up o 30 w . % may lead o
high alues in E ha exhibi a con inuous inc ease wi h CF/GL a io, bu lead
o pa icula ly high alues in εmax ha become less impo an o he highes

Resul s
289
CF/GL a io s udied. The la e e ec is so impo an in his case ha
oughness ollows he same e olu ion. The inal dec ease in εmax obse ed in
Figu e 5.4-21 and Figu e 5.4-22 is p obably because he specimen becomes
b i le as a consequence o he high amoun o polyme (CF+PCL).
I is also wo h men ioning ha a compa ison o he elas ic bending
modulus (E’) and he Young’s modulus (E) in Figu e 5.4-17 and Figu e 5.4-20,
espec i ely, pu o wa d he simila beha iou o bo h pa ame e s. In ac ,
bo h pa ame e s e lec he con ibu ion o CF p o ein and PCL o he elas ic
esponse a small s ain unde bending o uniaxial ension de o ma ions. All
hese esul s e eal ha bo h polyme s play a signi ican ole on he elas ic
p ope ies. In o de o assess he con ibu ion o each polyme (CF and PCL) a
compa ison be ween wo sys ems con aining he same polyme /GL a io
(40/30/30 and 60/30/10) may be ca ied ou . I is appa en ha he PCL yields
highe con ibu ion o he elas ic esponse, pa icula ly unde uniaxial ensile
es s. This dominan con ibu ion o PCL can be ex apola ed o he plas ic
de o ma ion egion since he 40/30/30 specimen shows much highe alue in
εmax. The oughness also pu o wa d his e ec .
X-Ray Di ac ion (XRD)
Figu e 5.4-23 shows he X- ay di ac ion spec a o he e e ence sys em
and sys ems con aining 10 w . % PCL.
This igu e e eals he cha ac e is ic pa e n o PCL in i s c ys alline
s uc u e wi h he well-de eloped peaks a 2θ = 21.5 ⁰ and 23.7 ⁰ in
acco dance wi h Ve uccio e al. (2009) ha also epo ed a shoulde peak a
abou 22.0 ⁰. A u he peak may be also obse ed a 29.7 ⁰, showing much
lowe in ensi y.
Resul s
290
10 15 20 25 30
0
50
100
150
200
250
2(Deg ee)
In ensi y (Coun s)
Symbol Sys em
CF/GL/PCL
(70/30/0)
CF/GL/PCL
(65/25/10)
CF/GL/PCL
(63/27/10)
CF/GL/PCL
(60/30/10)
Figu e 5.4-23: XRD o he e e ence sys em (70/30/0) and o CF/GL/PCL p obes a
cons an PCL concen a ion: 10 w . % o PCL: (65/25/10), (63/27/10) and (60/30/10)
As o he e e ence PCL- ee specimen, he di ac ion spec um displays
a b oad peak ha un o una ely is loca ed a he same posi ion o he wo
main peaks o PCL. This igu e also shows how he inco po a ion o PCL up o
10 w . % does no lead o any modi ica ion in he loca ion (2ϴ alue) o he
PCL peaks, bu a p og essi e b oadening e ec akes place o bo h peaks as
he p o ein con en inc eases. Acco ding o Unga (2004), X- ay di ac ion
peaks b oaden when he c ys al la ice becomes impe ec . Peaks b oaden
ei he when c ys alli es become smalle han abou a mic ome e o i la ice
de ec s a e p esen in la ge enough amoun and he e a e wo impo an
causes: size and s ain b oadening. The e o e, in acco dance wi h his
s a emen , he esul s ob ained o composi es con aining 10 w . % PCL
sugges ha CF p o ein ei he inhibi s he de elopmen o he PCL c ys alline
phase o a ou s he p esence o la ice de ec s.
Resul s
291
Figu e 5.4-24 shows he X- ay di ac ion spec a o sys ems con aining
30 w . % PCL: (55/15/30), (49/21/30) and (40/30/30).
15 20 25 30 35
0
200
400
600
800
1000
1200
1400 Symbol Sys en
PCL
CF/GL/PCL
(55/15/30)
CF/GL/PCL
(49/21/30)
CF/GL/PCL
(40/30/30)
In ensi y (Coun s)
2(Deg ee)
Figu e 5.4-24: XRD o p obes a cons an PCL concen a ion: 30 w . % (55/15/30),
(49/21/30) and (40/30/30)
This igu e shows how he di ac ion spec a o he composi es
con aining 30 w . % PCL nei he yield any pa icula modi ica ion in he
loca ion (2ϴ alue) o he PCL peaks. This ac means ha he in e plana
dis ance (dhkl) also emains unal e ed, and as a consequence, he la ice
s uc u e does no su e any change, ega dless o he p o ein con en o he
composi e specimen. These esul s sugges ha amo phous sec ions a e
esponsible o he p o ein-polyme sys em compa ibili y. Con e sely, i can
be obse ed ha an inc ease in he o al amoun o syn he ic polyme leads
o emain unal e ed c ys alline sec ions. I is also wo h poin ing ou ha he
c ys alline phase o he PCL is well de eloped o composi es con aining 30 w .
% PCL, which sugges s ha he selec ed p ocessing condi ions do no a ec o
Resul s
292
he PCL c ys alline mo phology. In addi ion, hese laye s do no exhibi any
b oadening e ec (nei he size no s ain), hus indica ing ha he
de elopmen o PCL c ys alline phase is p ese ed a e p ocessing, which may
well be ela ed o he imp o emen o mechanical p ope ies o hese
composi es con aining high pe cen age o PCL.
CF and CF2L p o ein-based bioplas ic
compa ison
In o de o compa e bo h p o ein sys ems (CF and CF2L) he a io o
p o ein-con en o plas icise was kep cons an .
Then, i he p o ein con en o he CF concen a e is 64.2 ± 0.9 w . %, he
o al amoun o p o eins in he bioplas ic p obes is abou 45 w . %. Thus, i he
p o ein con en o he CF2L sys em is 78.6 ± 0.9 w . %, he a io
p o ein/plas icise should be 60/40.
Fo his eason, he sys ems compa ed we e: CF/GL (70/30) and CF2L/GL
(60/40), being all injec ion condi ions o he new sys em ha one selec ed
om he p e ious s udy o CF p o ein concen a e.
Dynamic mechanical analysis and ensile es we e pe o med o
cha ac e ise he mechanical esponse o he new bioplas ic.
Dynamic Mechanical analysis
Figu e 5.4-25 shows a compa ison be ween sys ems con aining di e en
c ay ish powde (CF and CF2L) a he same p o ein/plas icise a io:
Resul s
293
-40 -20 0 20 40 60 80 100 120 140
104
105
106
107
108
109
CF2L/GL (60/40)
CF/GL (70/30)
E' / (Pa)
Tempe a u e (ºC)
Figu e 5.4-25: DMTA o he e e ence sys em CF/GL (70/30) and he sys em wi h
CF2L p o ein concen a e CF2L/GL (60/40).
As may be obse ed, CF-based sys em has highe elas ic modulus in he
o e all empe a u e in e al s udied. Howe e , bo h bio-based plas ic sys ems
(CF and CF2L), exhibi simila beha iou . The inc ease o empe a u e leads o
dec ease he elas ic modulus, un il c.a. 100 oC whe e ce ain he mose ing
po en ial seems o appea . The d ama ic dec ease o elas ic modulus obse ed
o CF/GL (70/30) p obes was no ound o CF2L/GL (60/40) p obes, howe e
elas ic modulus o CF/GL (70/30) is ne e below CF2L/GL (60/40) modulus.
Uniaxial ensile s eng h
Figu e 5.4-26 shows he ensile s eng h measu emen s o sys ems CF
and CF2L a he same p o ein/plas icise a io (70/30 and 60/40, espec i ely):

Resul s
294
0.00 0.01 0.02 0.03
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
S ess (MPa)
S ain (mm/mm)
CF2L/GL 60/40
CF/GL 70/30
Figu e 5.4-26: Tensile es o sys ems CF/GL and CF2L/GL a he same
p o ein/plas icise a io (70/30 and 60/40, espec i ely)
Pa ame e s ob ained om hese measu emen s a e shown in Table 5.4-3:
Sys em
Elonga ion a b eak (%)
Max. s ess (MPa)
E (MPa)
CF/GL (70/30)
CF2L/GL (60/40)
1.9 ± 0.2
2.9 ± 0.3
0.60 ± 0.12
0.62 ± 0.15
62.2 ± 4.5
38.4 ± 5.1
Table 5.4-3: Pa ame e s om ensile s eng h measu emen s o CF/GL (70/30) and
CF2L/GL (60/40) sys ems.
Resul s om ensile es s show ha bo h sys ems ha e compa able
maximum s ess alue, howe e CF p obes a e mo e igid, and as consequence
hese p obes exhibi lowe elonga ion a b eak and highe Young’s modulus.
Bo h p obes ha e compa able pa ame e s.
Resul s
295
A la ida
Po odos esos momen os que me has dado,
Po es e ciclo que no es aba p og amado, pe o que elegí sin sabe a dónde
llega ía ¿Qué hubie a sido de mí sin us enseñanzas?
297
6. Conclusions
Conclusions
304
9. The highes an ioxidan ac i i y was ob ained agains ABTS and he
lowes when FC was used, since his eagen is speci ic o phenol compounds.
No pa icula in luence o pH was ound o DPPH o FC ac i i y. Howe e , pH
exe s a d ama ic inc ease on he ac i i y agains ABTS (a ound one decade).
In gene al hyd olysa es sys ems exhibi a highe an ioxidan ac i i y excep
agains DPW,. This beha iou is ema kable o ABTS a pH 8.0 whe e
an ioxidan ac i i y eaches wice he alue o CF2L. Once again, a high
deg ee o hyd olysis is no desi able, because an ioxidan ac i i y dec eases.

Conclusions
305
6.4. Conclusions om C ay ish bioplas ic
10. F om he expe imen al esul s, i may be concluded ha moni o ing
he o que o e mixing o p o ein-based lou , addi i es and plas icize i is
use ul o selec he mo e sui able condi ions (e.g. mixing ime and
o mula ion) in e ms o ene gy e iciency. The addi ion o educing agen (BS
o SS), dena u ing agen (U) o c osslinking p omo e (LC), always yields an
inc ease in ene gy e iciency a he mixing s age, leading o a ema kable
educ ion in he linea iscoelas ic p ope ies o blends, which is also
impo an o selec sui able ope a ion condi ions o injec ion moulding
p ocessing.
11. As o bioplas ics, he addi i e de eloping a g ea e e ec on
mechanical p ope ies is LC, which p o ides specimens showing a highe alue
o he Young’s modulus, as well as a emnan he mose ing po en ial o
u he p ocessing a high empe a u e. Howe e , i is he maximum
elonga ion he p ope y ha is ema kably enhanced by inc easing he
he mose ing empe a u e, which akes place a he expense o he Young’s
modulus. The maximum s ess inc ease only when he empe a u e o he
mould inc ease.
12. The combina ion o echniques such as mixing heology o CF/GL/PCL,
con olling o que and empe a u e p o iles, and DSC is e y use ul o
selec ing sui able injec ion moulding pa ame e s o p ocessing blends.
13. CF/GL/PCL biocomposi es show a ema kable enhancemen in
mechanical p ope ies as compa ed o CF/GL bioplas ics, e en when
c ys alline s uc u e emains unal e ed. Fu he mo e, he p o ein/plas icise
a io also plays a ele an ole. Thus, bo h polyme s play a signi ican ole on
he elas ic p ope ies. Howe e , he PCL yields a dominan con ibu ion o he
Conclusions
306
elas ic esponse, pa icula ly unde uniaxial ensile es s, and con e a highe
abili y o abso b ene gy be o e up u e.
14. Finally, he easibili y o designing enewable and biodeg adable
composi es ha e been demons a ed, which may be ega ded as an
al e na i e o con en ional plas ic ma e ials, con aining an impo an amoun
o CF, he eby inding alue-added applica ions o hese low alued by-
p oduc s o he c ay ish indus y.
307
7. Re e ences.
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Appendix
329
8. Appendix
T¼Z
ε
max
0
sðεÞ$dε(1)
Rega ding he ensile p ope ies ob ained om ensile es s
applied o CF/GL, and CF/GL/PCL biocomposi es, Fig. 6BeD show he
alues o he Young's Modulus, s ain a b eak and oughness,
espec i ely. As may be obse ed, highe alues in E0and especially
in ε
max
and oughness a e ob ained by inc easing PCL om 0 o 30%.
Mo eo e , an inc ease in he CF/GL a io yields a di e en
beha iou depending on PCL con en . Those specimens con aining
10 w .% PCL display a maximum alue in E as well as a minimum
alue in ε
max
. As a esul , a mode a e inc ease in oughness akes
place. I should be also aken in o accoun ha an inc ease in he CF/
GL a io also in ol es a dec ease in he PCL/CF a io. These esul s
sugges ha bo h a ios exe opposi e e ec s, whe e he dominan
e ec seems o be he o me a low CF con en and he la e a
high CF con en . This balance can explain he occu ence o he
maximum in E and minimum in ε
max
.
On he o he hand, an inc ease in PCL con en up o 30 w .% may
lead o high alues in E ha exhibi a con inuous inc ease wi h CF/
GL a io, bu lead o pa icula ly high alues in ε
max
ha become
less impo an o he highes CF/GL a io s udied. The la e e ec
is so impo an in his case ha oughness ollows he same e o-
lu ion. The final dec ease in ε
max
obse ed in Fig. 6C and D is
p obably because he specimen becomes b i le as a consequence o
he high amoun o polyme (CF þPCL).
I is also wo h men ioning ha a compa ison o he elas ic
bending modulus (E0) and he Young's modulus (E) in Figs. 4A and
6B, espec i ely, pu o wa d he simila beha iou o bo h pa-
ame e s. In ac , bo h pa ame e s eflec he con ibu ion o CF
p o ein and PCL o he elas ic esponse a small s ain unde
bending o uniaxial ension de o ma ions. All hese esul s e eal
ha bo h polyme s play a significan ole on he elas ic p ope ies.
In o de o assess he con ibu ion o each polyme (CF and PCL) a
compa ison be ween wo sys ems con aining he same polyme /GL
a io (40/30/30 and 60/30/10) may be ca ied ou . I is appa en ha
he PCL yields highe con ibu ion o he elas ic esponse, pa ic-
ula ly unde uniaxial ensile es s. This dominan con ibu ion o
PCL can be ex apola ed o he plas ic de o ma ion egion since he
40/30/30 specimen shows much highe alue in ε
max
. The ough-
ness also pu o wa d his e ec .
4. Concluding ema ks
F om he expe imen al esul s, i may be concluded ha mixing
p ocess o p o ein-based flou , plas icise and syn he ic polyme
can be con olled by moni o ing he o que and empe a u e p o-
files in o de o selec he mos sui able mixing ime. The combi-
na ion o echniques such as mixing heology and DSC a e e y
use ul o selec ing sui able injec ion moulding pa ame e s o
p ocessing blends. In his way, i could be claimed ha a bioplas ic
exhibi ing desi able p ope ies can be made by means o injec ion
moulding, using a sui able o mula ion (c ayfish flou , glyce ol and
PCL) and selec ing p ope he momechanical p ocessing condi ions
(injec ion p essu e, empe a u e and esidence ime in he p e-
injec ion chambe and empe a u e in he mould).
F om he mechanical cha ac e isa ion o he CF/GL/PCL bio-
composi es, i can be poin ed ou he ema kable enhancemen in
mechanical p ope ies ob ained o PCL con aining sys ems e en
when c ys alline s uc u e emains unal e ed. Fu he mo e, as
ega ds he biocomposi e o mula ion, no only he con ibu ion o
PCL is impo an bu also he p o ein/plas icise a io plays a ole.
Thus, bo h polyme s play a significan ole on he elas ic p ope ies,
howe e , he PCL yields a dominan con ibu ion o he elas ic
esponse, pa icula ly unde uniaxial ensile es s and con e a
highe abili y o abso b ene gy be o e up u e.
Fig. 6. Resul s om uniaxial Tensile S eng h measu emen s o he specimens s udied 70/30/0, 65/25/10, 63/27/10, 60/30/10, 55/15/30, 49/21/30 and 40/30/30: (A) Tensile s ess-
s ain cu es (only o specimens a cons an CF/GL a io: 70/30/0, 63/27/10 and 49/21/30); (B) Young's modulus (E); (C) s ain a b eak (Ɛ
Max
); (D) Toughness (T).
M. F
elix e al. / Composi es Pa B 78 (2015) 291e297296

The p esen wo k demons a es he easibili y o designing
enewable and biodeg adable composi es, which may be ega ded
as an al e na i e o con en ional plas ic ma e ials, con aining an
impo an amoun o CF, he eby finding alue-added applica ions
o hese by-p oduc s o he c ayfish indus y.
Acknowledgemen s
This wo k is pa o a esea ch p ojec sponso ed by Andalousian
Go e nmen , (Spain) (p ojec TEP-6134) and by “Minis e io de
Economía y Compe i i idad” om Spanish Go e nmen
(Re . MAT2011-29275-C02-02/01). The au ho s g a e ully acknowl-
edge hei financial suppo . The au ho s also acknowledge o he
Mic oanalysis Se ice, X-Ray and Func ional Cha ac e isa ion Se ice
(CITIUS-Uni e sidad de Se illa) o p o iding ull access and assis-
ance o he LECOeCHNSe932, D8 Disco e (B uke ) equipmen and
DSC Q20 Calo ime y (TA ins umen s), espec i ely.
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679
Resea ch A icle
Recei ed: 3 Feb ua y 2014 Re ised: 21 Ap il 2014 Accep ed a icle published: 6 June 2014 Published online in Wiley Online Lib a y: 25 June 2014
(wileyonlinelib a y.com) DOI 10.1002/js a.6747
De elopmen o c ayfish bio-based plas ic
ma e ials p ocessed by small-scale injec ion
moulding
Manuel Felix,*Albe o Rome o, Felipe Co dobes and An onio Gue e o
Abs ac
BACKGROUND: P o ein has been in es iga ed as a sou ce o biodeg adable polyme ic ma e ials. This wo k e alua es he de el-
opmen o plas ic ma e ials based on c ayfish and glyce ol blends, p ocessed by injec ion moulding, as a ully biodeg adable
al e na i e o con en ional polyme -based plas ics. The effec o diffe en addi i es, namely sodium sulfi e o bisulfi e as educ-
ing agen s, u ea as dena u ing agen and L-cys eine as c oss-linking agen , is also analysed.
RESULTS: The inco po a ion o any addi i e always yields an inc ease in ene gy efficiency a he mixing s age, bu i s effec
on he mechanical p ope ies o he bioplas ics is no so clea , and e en dampened. The addi i e de eloping a g ea e effec
is L-cys eine, showing highe Young’s modulus alues and exhibi ing a emnan he mose ing po en ial. Thus, p ocessing a
highe empe a u e yields a ema kable inc ease in ex ensibili y.
CONCLUSION: This wo k illus a es he easibili y o c ayfish-based g een biodeg adable plas ics, he eby con ibu ing o he
sea ch o po en ial alue-added applica ions o his by-p oduc .
© 2014 Socie y o Chemical Indus y
Keywo ds: bioplas ic; c ayfish p o ein; mixing blends; heology; ensile s eng h es
INTRODUCTION
The eshwa e ed-swamp c ayfish (P ocamba us cla kii)was
in oduced in o Eu ope in he ea ly 1960s. Since hen, his species
has unde gone a as widesp ead g ow h due o i s esis ance
o ungal disease as well as o a ou able wea he condi ions,
abundan ood and a lack o p eda o s.1This ac has d i en he
de elopmen o a s ong local c ayfish indus y a he ma shes
o he Guadalqui i Ri e in Spain. This de elopmen has also led
o he gene a ion o a la ge amoun o c ayfish su pluses, as is
equen ly he case in he fish and shellfish indus y.2Acco ding
o ecen es ima ions by he Food and Ag icul u e O ganiza ion
he o al wo ld ou pu o fish ep esen ed 156 million onnes
in 2011. Un o una ely, a high p opo ion o his amoun is no
e en ually used o human ood consump ion. Thus, acco ding
o he USDA, up o 45% o fish and shellfish ha en e s he USA
e ail ood ma ke is no ea en and, as a consequence, ends as
was es. The e o e, a sea ch o any alue-added applica ion o
hese su pluses is becoming a eal p io i y.
Wi h ega d o c ayfish su pluses, some applica ions based
on he unc ional p ope ies o c ayfish p o ein ac ion ha e
p e iously been assessed. Thus, in e acial p ope ies o c ayfish
p o ein isola e (CFPI) ha e been s udied3–5in o de o add ess he
good pe o mance o c ayfish p o ein in emulsion s abilisa ion.6,7
The mal p ope ies o c ayfish flou 8and CFPI in aqueous solu-
ion ha e also been s udied in o de o assess he mally induced
enhancemen o emulsion s abili y9as well as he po en ial o
c ayfish in he manu ac u e o su imi-like p oduc s based on i s
abili y o o m a gel.10,11
A cu en ly a ac i e way o alo ise hese by-p oduc s is
h ough hei use as enewable esou ces in he manu ac u e o
‘g een ma e ials’, eplacing difficul - o-deg ade plas ic ma e ials
made om oil-based syn he ic polyme s. Nowadays, some impo -
an applica ions o bioplas ics a e beginning o eme ge in he
a eas o packaging, ood p oduc ion, pha maceu ics, elec on-
ics, au omo i e indus y and biomedicine. Thus, among o he
applica ions bioplas ics can be used in ood packaging, ui
coa ing, encapsula ion, ex iles, abso ben ma e ials o issue
enginee ing.12–14 This wide a ie y o po en ial applica ions
allows us o en isage an inc easing use o biobased-plas ic ma e-
ials. Thus, acco ding o Eu opean Bioplas ics, he p oduc ion
capaci y o bioplas ics is p edic ed o inc ease om app oxi-
ma ely 700 000 ons in 2010 o 1.7 M ons by 2015.12 Howe e ,
like o he bio-based inno a ions, bioplas ics ha e s uggled o
achie e ma ke sha e such ha , a p esen , bioplas ics cons i u e
less han 0.5% o wo ld plas ics consump ion.15 The e o e, i is s ill
necessa y o in ensi y he effo s in esea ch and de elopmen ,
and in inno a ion in his field.
Some biopolyme s can di ec ly eplace syn he ically de i ed
ma e ials in adi ional applica ions o hey simply possess unique
∗Co espondence o: Manuel Felix, Depa amen o de Ingenie ía Química, Uni-
e sidad de Se illa, Facul ad de Química, 41012 Se ille, Spain.
E-mail: [email p o ec ed]
Depa amen o de Ingenie ía Química, Uni e sidad de Se illa, Facul ad de
Química, 41012 Se ille, Spain
J Sci Food Ag ic 2015; 95: 679–687 www.soci.o g © 2014 Socie y o Chemical Indus y
680
www.soci.o g M Felix e al.
p ope ies ha could open up a ange o new comme cial oppo -
uni ies. In any case, i is essen ial ha bio-based ma e ials exhibi
sui able physico-chemical p ope ies. In his sense, cellulose,
s a ch, polysaccha ides and p o ein ha e become inc easingly
compe i i e in ecen yea s as subs i u es o pe ochemicals, in
iew o he inc ease in oil p oduc ion cos s. Bo h en i onmen al
and economic ac o s a e expec ed o en ail he de elopmen o
new plas ic ma e ials such as hose using by-p oduc s wi h high
p o ein con en .16 In his espec , p o eins a e excep ionally e sa-
ile ma e ials, bo h in he sou ces om which hey can be ob ained
and in he wide a ie y o possible modifica ions, which can be
help ul in ailo ing hei p ope ies o he pa icula equi emen s
o a specific applica ion. P o eins p esen significan ad an ages
in ha hey a e de i ed om a sus ainable esou ce and can
be p ocessed in much he same way as con en ional syn he ic
polyme s.13 Howe e , p o eins a e gene ally mixed wi h a plas-
icise in o de o educe in e molecula o ces among polyme
chains, inc easing mobili y and educing he glass ansi ion.17
T adi ionally, p o ein films18–20 a e p ocessed by a cas ing
me hod; howe e , classical polyme p ocessing echniques (com-
p ession moulding o ex usion) a e being inc easingly used in his
field.21–24 Among hem, injec ion moulding is a ai ly a ac i e
ope a ion ha has no ecei ed due a en ion ye . Thus, s udies
on p o ein-based biodeg adable polyme ic ma e ials p ocessed
by injec ion moulding a e sca ce.25,26
Typically in his p ocess, polyme ic ma e ials a e subjec ed o
sui able he mal condi ions, being injec ed a high p essu e in o
he mould ca i y. Op imisa ion o p ocessing condi ions is essen-
ial o achie e he desi e p ope ies o he final p oduc . This
is pa icula ly ele an in p o ein-based ma e ials ha equi e
he moplas ic mixing wi h a p ope plas icise bu benefi om
a p edominan he mose cha ac e upon injec ion moulding.26
Besides, addi i es such as educing agen s may be help ul in o de
o educe he a e age molecula weigh o p o ein agg ega es,
hus acili a ing bo h mixing and moulding p ocesses. Thus, some
au ho s s udied he influence o educing agen s on he p ope -
ies o he mo-moulded whea glu en bioplas ics,27 soy p o ein
isola e,28–30 s a ch31 o flax fib es.32 The effec o adding a dena-
u ing agen , such as u ea has also been analysed on bioplas ics
p ocessed by ex usion33 o comp ession moulding.34–36 As o he
use o L-cys eine as c oss-linking agen , Sun e al.37 ha e ecen ly
e alua ed i s pe o mance on he mo-moulded glu en-based plas-
ics. Howe e , no in o ma ion abou c ayfish-based bioplas ics
wi h o wi hou educing, dena u ing o c oss-linke agen s has
been ound.
The o e all objec i e is o e alua e he po en ial de elopmen
o plas icised c ayfish bio-based plas ic ma e ials by means o a
con en ional and highly e sa ile polyme p ocessing echnique,
such as injec ion moulding, as an al e na i e o moulded ma e ials
based on polyme s de i ed om ossil uels. A u he objec i e is
o analyse he effec o using diffe en addi i es on he p ope ies
o c ayfish-based p oduc s, using glyce ol as he plas icise . The
addi i es assessed in his s udy we e sodium sulfi e o bisulfi e
as educing agen s, u ea as dena u ing agen and L-cys eine as
c oss-linking agen . A small-scale plunge - ype injec ion moulding
machine was used in his s udy o ob ain c ayfish-based specimens
om c ayfish/glyce ol/addi i e blends, p e iously mixed by means
o a mixing heome e ha allows he o que and empe a u e o
be eco ded du ing mixing. Rheological and diffe en ial scanning
calo ime y measu emen s o hese blends we e also ca ied ou
in o de o ob ain in o ma ion ha may be used in he selec ion o
sui able p ocessing pa ame e s o injec ion moulding ope a ions
(e.g. empe a u e and esidence ime in he p e-injec ion cylinde
as well as he empe a u e o he mould).
EXPERIMENTAL
Ma e ials
C ayfish flou was ob ained om ALFOCAN S.A. (Isla Mayo , Se ille,
Spain). The p o ein con en o he c ayfish flou , de e mined
in quad uplica e as % N ×6.25 using a LECO CHNS-932 ni o-
gen mic o analyse (Leco Co po a ion, S Joseph, MI, USA), was
642 ±9gkg
−1. Glyce ol (used as plas icise ) and addi i es (sodium
sulfi e, sodium bisulfi e, u ea and L-cys eine) we e pu chased om
Pan eac Química, S.A. (Ba celona, Spain).
F ee sul hyd yl g oups
F ee sul hyd yl g oups o p o ein samples we e de e mined
using he me hod de eloped by Be e idge e al.38 Samples
we e suspended (10 g L−1) in buffe con aining 0.086 mol L−1
T is–HCl, 0.09 mol L−1glycine, 4 mmol L−1EDTA and 8 mol L−1
u ea, a pH 8. Dispe sions we e s i ed a 25 ∘C du ing 10 min a
500 pm in a he momixe and hen cen i uged a 15 000 ×g
(10 min, 10 ∘C). Supe na an was incuba ed wi h Ellman’s
eagen [5,5′-di hiobis-(2-ni obenzoic acid) (DTNB); 4 g DTNB L−1
me hanol]. Abso bance a 412 nm was measu ed in a Genesis-20
spec opho ome e (The mo Scien ific, Wal ham, MA, USA). The
mola ex inc ion coefficien o 3- hio-6-ni obenzoa e (TNB;
13 600 M−1cm−1) was used. P o ein concen a ion o ex ac s was
de e mined by he B ad o d me hod.
Sample p epa a ion
Blends wi h diffe en c ayfish flou /glyce ol (CF/GL) a ios we e
manu ac u ed by a he momechanical p ocedu e which consis ed
o wo s ages. Fi s ly, selec ed blends con aining 700 g kg−1c ay-
fish flou and 300 g kg−1glyce ol (deno ed 70/30) we e mixed
in a wo-blade coun e - o a ing ba ch mixe Haake Polylab QC
(The moHaake, Ka ls uhe, Ge many) a 25 ∘Cand50 pm o 60
o 20 min, moni o ing he o que and empe a u e du ing mix-
ing. Secondly, he dough-like ma e ials ob ained a e mixing we e
subsequen ly p ocessed by injec ion moulding using a MiniJe
Pis on Injec ion Molding Sys em II (The moHaake) o ob ain bio-
plas ic specimens. Two moulds we e used o p epa e wo ype
o specimens: (1) a 60 ×10 ×1 mm ec angula -shaped specimen,
o be used o bo h dynamic mechanical empe a u e analysis
(DMTA) expe imen s and anspa ency measu emen s, and (2) a
dumb-bell- ype specimen defined by ISO 527–2:1993 o de e -
mining he ensile p ope ies o plas ics.
Cha ac e isa ion o blends
The mos sui able p ocessing a iables such as empe a u es in he
p e-injec ion cylinde o in he mould we e selec ed a e pe o m-
ing empe a u e amp es s and diffe en ial scanning calo ime y
measu emen s ( esul s no shown).
Rheological measu emen s
Dough-like ma e ials we e cha ac e ised by small ampli ude oscil-
la o y shea measu emen s, using a con olled-s ain heome e ,
in o de o selec he op imum condi ions o injec ion mould-
ing: (Ma s II om Haake, Ka ls uhe, Ge many). The geome y used
has been a pla e and pla e geome y (diame e , 25 mm) wi h a
ough su ace and a gap be ween pla es o 1 mm. Low iscosi y
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Dow Co ning 200 fluid was used as sealan o a oid sample d ying.
S ain sweep small ampli ude oscilla o y shea es s we e also
pe o med in o de o es ablish he linea isco-elas ici y ange.
Tempe a u e amp es s we e ca ied ou a 5 ∘Cmin
−1 om 20
o 100 ∘C and ime sweep es s we e pe o med o 1800 s a a
selec ed cons an empe a u e. Linea isco-elas ic p ope ies (G′,
G′′) we e moni o ed a a cons an equency o 6.28 ad s−1.All he
sys ems s udied had he same he mo- heological his o y be o e
pe o ming any heological es .
Cha ac e isa ion o bioplas ics
Dynamic mechanical empe a u e analysis
DMTA es s we e ca ied ou wi h a RSA3 (TA Ins umen s, New
Cas le, DE, USA), on ec angula p obes using dual can ile e bend-
ing. All he expe imen s we e ca ied ou a cons an equency
(6.28 ad s−1) and s ain (be ween 0.01 and 0.3%, wi hin he linea
isco-elas ic egion). The selec ed hea ing a e was 3 ∘Cmin
−1.All
he samples we e coa ed wi h Dow Co ning high acuum g ease
o a oid wa e loss.
Tensile s eng h measu emen s
Tensile es s we e pe o med by using an Insigh 10 kN Elec ome-
chanical Tes ing Sys em (MTS, Eden P ai ie, MN, USA), acco ding o
ISO 527–2:1993 o ensile p ope ies o plas ics. Tensile s ess and
elonga ion a b eak we e e alua ed om a leas h ee duplica es
o each p oduc using ype IV p obes and an ex ensional a e o
100 mm min−1a oom empe a u e.
S a is ical analysis
A leas h ee eplica es o each measu emen we e ca ied ou .
S a is ical analyses we e pe o med using a - es and one-way
analysis o a iance (ANOVA, P<0.05) by using he s a is ical
package SPSS 18 (SPSS, Chicago, IL, USA). S anda d de ia ions om
some selec ed pa ame e s we e calcula ed.
RESULTS AND DISCUSSION
The moplas ic mixing o blends
Figu e 1 shows bo h o que and empe a u e p ofiles as a unc ion
o mixing ime o blends ob ained a diffe en CF/GL a ios, as
well as hei isual appea ance a e mixing. These esul s show
he ema kable dependence o hese pa ame e s on he CF/GL
a io. Thus, a apid inc ease in o que up o a maximum alue akes
place o he sys em ha ing less amoun o plas icise , deno ed
as 80/20, which ep esen s he CF/GL weigh a io. This e olu ion
is eadily ollowed by an asymp o ic dec ease owa ds a pla eau
alue. In con as , he sys em 70/30 shows a mode a e g ow h
in o que showing no maximum alue bu a slow endency o a
pla eau alue. The p ofile o he sys em 60/40 only shows an ini ial
inc ease in o que, al hough e y sligh , being domina ed by a
cons an o que alue o e mixing ime. In gene al, he e olu ion
o empe a u e o e ime is e y simila o he o que p ofile
whe e an inc ease akes place excep ing o he lowes CF/GL a io.
Bo h inc eases in o que and empe a u e may be a ibu ed o
shea -induced exo he mic eac ions, by S—S bonds o ma ion,
de eloped du ing he mixing p ocess.
As a consequence o he abo e-men ioned diffe ences in o que
p ofile, he specific mechanical ene gy (SME) inpu o mixing is
also qui e diffe en . This cha ac e is ic pa ame e o mixing may
be defined as ollows:39
SME =𝜔
m∫ mix
0
M( )d (1)
whe e 𝜔(in ad s−1) is he mixing speed, m(in g) is he sample mass,
M( )(inNm)is he o queand mix (in s) is he mixing ime. The
alues o he SME pa ame e o hese h ee sys ems a e included
in Fig. 1. A ema kable inc ease in his pa ame e can be obse ed
wi h he CF/GL a io. This effec akes also place a he highe
concen a ions in spi e o he ac ha he pla eau o que alues
a e ela i ely close o each o he .
F om he isual appea ance obse ed in Fig. 1, sys em 80/20
does no seem o con ain enough amoun o plas icise o ob ain
an easy- o-handle ma e ial, gi ing ise o a g anula ed powde
ins ead o a homogeneous dough-like blend. In ac , his is he
sys em displaying he highes o que alues, which co espond
o he highes heological consis ency. On he o he hand, he
highes amoun o glyce ol in he 60/40 sys em p o ides a nea
dough-like appea ance o he blend. This is also consis en wi h
he lowes o que alues ob ained (associa ed wi h a lowe con-
sis ency) a e he mixing p ocess. The 70/30 sys em shows in e -
media e beha iou be ween bo h limi s, al hough i is close o
he mos concen a ed p o ein-based blend. In he p esen s udy,
he 80/20 sys em has been disca ded because o he high ene gy
Figu e 1. Mixing o que and empe a u e p ofiles and images o c ayfish flou /glyce ol (CF/GL) sys ems (80/20, 70/30 and 60/40).
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AB
Figu e 2. E olu ion o mixing o que (A) and empe a u e (B) as a unc ion o mixing ime o addi i e-con aining sys em (sodium sulfi e, u ea and
L-cys eine). SS, sodium sulfi e; U, u ea; LC, L-cys eine.
inpu equi ed o mixing. Mo eo e , his sys em has p o en o
be difficul o injec ( esul s no shown). On he o he hand, sys-
ems con aining a low p o ein/glyce ol a io may be injec ed easily
bu he final bioplas ic ma e ials end o exude he excess glyc-
e ol. Exuda ion is a well-known phenomenon desc ibed in he
polyme –plas icise li e a u e and should be a oided in o de o
p e en con amina ion o he su ounding ma e ials (e.g. in ood
packaging).40 This seems o be he case o he 60/40 sys em. As a
esul , he 70/30 sys em was selec ed as he mos sui able o ob ain
a homogeneous and easily injec able CF/GL blend.
Figu e 2 shows he e olu ion o o que (Fig. 2A) and empe a u e
(Fig. 2B) as a unc ion o ime o diffe en 70/30 CF/GL blends p e-
pa ed using sodium sulfi e as educing agen , u ea as dena u ing
agen , o L-cys eine as c oss-linking p omo e a diffe en concen-
a ions (3 and 30 g kg−1p o ein o sodium sulfi e and u ea o 1
and 3 g kg−1 o L-cys eine).
These esul s gene ally show ha any o he addi i es used
induces an an icipa ion o he o que p ofile, pa icula ly a he
highes addi i e concen a ions. No diffe ences in he o que
p ofile a e no iced a he wo diffe en concen a ions o sodium
bisulfi e (da a no shown). The highes amoun o addi i e (co -
esponding o sodium sulfi e o u ea) also yields lowe o que
pla eau alues, which may be conside ed beneficial o he p o-
cessabili y o blends, o example unde injec ion moulding.
Tempe a u e– ime p ofiles show he same ype o e olu ion o
all he addi i es and concen a ions in spi e o he ac ha an ai
s eam was used as a cooling fluid. No diffe ences a e ound in he
final alues o he highes sodium sulfi e o u ea con en in his
case. The only diffe ence ound be ween empe a u e and o que
p ofiles is ha he o me unde go a u he delay in ime, which
may be ela ed o he ai -cooling effec .
I is in e es ing o no ice ha al hough all he addi i es in
Fig. 2 led o an appa en an icipa ion in o que and empe a u e
p ofiles, diffe en mechanisms a e in ol ed depending on he
addi i e used. U ea is a widely used dena u ing agen ha induces
dis up ion o physical in e ac ions among p o ein molecules. On
he o he hand sodium sulfi e, ac ing as a educing agen , induces
dis up ion o S—S bonds. As a consequence, p o ein–plas icise
mixing is a ou ed in bo h cases esul ing in a as e kine ics
o e he addi i e- ee sys em. L-Cys eine is an amino acid ha
p o ides a ee sul hyd yl g oup/molecule and he e o e acili a es
o ma ion o S—S bonds wi h p o ein segmen s o o m cys ine
g oups.41 In his way, L-cys eine ini ia es he o ma ion o exo he -
mic S—S bonds, p og essi ely inc easing he empe a u e, which
leads o an icipa ion o bo h o que and empe a u e p ofiles. In
ac , his addi i e gi es ise o he as es an icipa ion o he same
addi i e con en . In addi ion, he highes empe a u es eached
by he L-cys eine-con aining blend sugges he occu ence o a
highe c oss-linking deg ee as compa ed o he o he sys ems.
I may be also no ed ha he sys em is no s ill p ope ly mixed
a imes sho e han ha ha co esponding o he maximum
o que. The e o e, as a gene al ule, he ange o mixing imes
o subsequen blend p ocessing is selec ed once he pla eau
o que alue is eached such ha a good deg ee o homogenei y
is assu ed.
Ano he in e es ing pa ame e o compa e diffe en mixing p o-
cesses is he ela i e ene gy inpu (REI), which is defined as ollows:
REI (%)=SMEmin (a,c)
SMEmin (0)×100 (2)
whe e SMEmin(0) and SMEmin(a,c) a e he SME alues a which he
o que eaches 95% o he maximum alue o he p ofile o he
addi i e ee blend and addi i e aa concen a ion c, espec i ely.
The e o e, REI is an index o he educ ion in ene gy inpu ela i e
o ha ha equi ed o mixing he addi i e ee blend.
The alues o SME20 and REI pa ame e s o all hese addi i es a e
shown in Fig. 3A and B, espec i ely. The SME alues o sodium
bisulfi e a e also included in his figu e. SME20 is he specific
mechanical ene gy supplied, as defined in Eqn (1), a e mixing o
20 min.
As may be obse ed in Fig. 3, an inc ease in addi i e concen-
a ion always leads o an appa en inc ease in he pa ame e
SME20, as a consequence o he abo e-men ioned an icipa ion o
he o que p ofile. I he same addi i e concen a ion is used, his
inc ease is pa icula ly ema kable o L-cys eine and, in any case,
is only mode a e when sodium bisulfi e is used.
The esul s ob ained wi h pa ame e REI confi m he ele an
effec o he addi ion o L-cys eine a cons an addi i e concen a-
ion. Thus, es ima ion endencies om he esul s ob ained indi-
ca e ha he addi i e/p o ein a io equi ed o achie e a 50%
REI would be 33 g kg−1 o u ea and 27 g kg−1 o sodium sulfi e,
whe eas a a io as low as 4.4 g kg−1wouldbe equi edbyusing
L-cys eine.
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A
B
Figu e 3. (A) Specific mechanical ene gy (SME) a e mixing o 20 min
and (B) educ ion in ene gy inpu (REI) o diffe en c ayfish flou /glyce ol
(CF/GL) blends (addi i e- ee, sodium bisulfi e, sodium sulfi e, u ea o
L-cys eine). BS, sodium bisulfi e; SS, sodium sulfi e; U, u ea; LC, L-cys eine.
Tempe a u e amps
Figu e 4 shows he dependence o linea isco-elas ic unc ions
on empe a u e o diffe en blends con aining 3 g addi i e kg−1
c ayfish p o ein. The addi i e ee blend is also displayed in his
figu e. All he addi i es s udied induce a educ ion in G′and G′′
in he expe imen al empe a u e ange. As o an 𝛿p ofiles, all
o hem show a simila elas ic-dominan beha iou cha ac e ised
by he p esence o a b oad dis ibu ion showing a maximum
alue. This maximum has been also ound a 70 ∘C by diffe en ial
scanning calo ime y measu emen s o he CF/GL sys em ( esul s
no shown), being associa ed o a glass-like ansi ion o he
p o ein ac ion. Mos o addi i e-con aining blends show his
smoo h he mal induced glass ansi ion in he same empe a u e
ange han he addi i e ee sys em (70–83 ∘C).Howe e ,ashi
in his e en owa ds highe alues (80–87 ∘C) akes place when
L-cys eine is used as addi i e.
I should be men ioned ha he cooling–se ing s age a e mix-
ing leads o no iceable diffe ences be ween he isco-elas ic p op-
e ies o addi i e- ee and addi i e-con aining CF/GL blends. Once
again, he addi i e showing he poo es effec is sodium bisulfi e.
Howe e , he cooling–se ing s age does no induce any appa en
diffe ence in G′o G′′ among he o he h ee addi i es (Fig. 4A),
in spi e o he ac ha he expec ed mechanism would be diffe -
en . Thus, sodium sulfi e is expec ed o educe he isco-elas ic
p ope ies o CF/GL blends by b eaking some disulfide bonds,
al hough he effec does no seem ake place a a high ex en .
A simila mechanism should explain he beha iou ound o
sodium bisulfi e-con aining blends, which becomes mo e impo -
an abo e 60 ∘C. In con as , u ea may show a double effec . Fi s ly,
as epo ed by Ve beek and Van den Be g,42 u ea may play he
ole o a plas icise a a concen a ion as low as ha used in his
s udy, no showing any c oss-linking effec . Secondly, u ea, as a
dena u ing agen , ends o inhibi he eco e y o physical in e -
ac ions o e he cooling–se ing s age. In ac , he la e effec
seems o be mo e ele an han he o me , as no diffe ences
in he an 𝛿peak can be de ec ed in Fig. 4B. Acco ding o his
figu e, he L-cys eine-con aining blend is he only one showing
he an 𝛿peak a highe empe a u e han he addi i e- ee sys-
em. This effec may be ela ed o a highe c oss-linking ex en ,
as p e iously men ioned (Fig. 2B). Howe e , his highe deg ee
o c oss-linking does no in ol e any inc ease in G′and G′′.On
he con a y, he esul s a e simila o hose shown by u ea- and
sodium sulfi e-con aining blends. An explana ion o his effec
should be ela ed o he inhibi ion o physical in e ac ions de el-
opmen o e he cooling–se ing s age.
In any case, he addi ion o any o he addi i es used leads o a
po en ial enhancemen o he moulding p ocessabili y o CF/GL
blends, whe e heological p ope ies a e ega ded as he key
AB
Figu e 4. (A) S o age modulus (G′) and loss modulus (G′′), and (B) loss angen ( an 𝛿), a 1 Hz and 5 ∘Cmin
−1, as a unc ion o empe a u e o diffe en
c ayfish flou /glyce ol (CF/GL) blends (addi i e- ee o 3 g kg−1sodium bisulfi e, sodium sulfi e, u ea o L-cys eine). BS, sodium bisulfi e; SS, sodium sulfi e;
U, u ea; LC, L-cys eine.
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Table 1. S anda d alues o injec ion moulding pa ame e s o he
p e-injec ion cylinde , injec ion and packing s age
Pa ame e T (∘C) P essu e (MPa) Time (s)
P e-injec ion cylinde 60 0.1 100a
Injec ion 60–100 0.1–50 <1
Packing s age 100 o 130 50 20
100 o 130 20 200a
aFo a sys em con aining L-cys eine, 130 ∘C and 600 s we e also used.
ac o . In o he wo ds, addi i es con ibu e o acili a e injec ion
o CF-based blends. Simila esul s we e ound by Pallos e al.43
o he mo o med whea glu en modified by educing agen s and
addi i es.
Injec ion moulding p ocess
Table 1 shows he condi ions selec ed o he injec ion mould-
ing p ocess o each o he blends s udied. P ocessing pa ame-
e s o he p e-injec ion cylinde a e selec ed aking in o accoun
ha he elas ic p ope ies o CF/GL blends should be educed
o some ex en ( ypically G′should be in he o de o 1 ×106Pa
o below). On he o he hand, he empe a u e should no be
inc eased excessi ely in o de o p e en he mally induced p o-
ein c oss-linking effec s be o e he injec ion s age. Fo he same
eason, he esidence ime in he cylinde should no be long.
Unde such p emises he pa ame e s selec ed o he fi s s age
ha e been 60 ∘C and 100 s.
As o he mould p ocessing condi ions, he empe a u e has o
be high enough o ensu e he no mal de elopmen o he mal
and p essu e-induced p o ein c oss-linking eac ions. On he o he
hand, exposu e o high empe a u es o a long ime ypically leads
o p o ein deg ada ion (i.e. ia Mailla d- ype eac ions). The e o e,
100 ∘C and 20 s ha e been selec ed as he moulding empe a u e
and ime, espec i ely. The injec ion p essu e selec ed in his
case has been 50 MPa. Once he blend has been injec ed in o he
mould, a u he s age is pe o med a he same empe a u e o
a esidence ime long enough (10 s) o allow o he de elopmen
o p o ein c oss-linking o achie e he final ne wo k s uc u e.
The esidence ime selec ed has been 200 s since no u he
enhancemen has been no ed by inc easing his pe iod.
Mechanical cha ac e isa ion o bioplas ics
Dynamic mechanical empe a u e analysis
Figu e 5 shows he alues o he elas ic modulus, E′, as a unc ion
o empe a u e ( om −30 ∘C o 140∘) ob ained om dynamic
mechanical analysis (DMA) measu emen s o addi i e- ee
CF/GL specimens and CF/GL p obes con aining 3 g kg−1addi i e.
Figu e 5A compa es he influence o diffe en addi i es (sodium
bisulfi e, sodium sulfi e, u ea and L-cys eine) o specimens
moulded a 100 ∘C o 200 s, whe eas Fig. 5B shows he effec o di -
e en moulding condi ions o L-cys eine-con aining specimens.
As may be obse ed in Fig. 5A, all he specimens show simila
p ofiles o E′andalso o E′′ (da a no shown), unde going a
ema kable dec ease wi h inc easing empe a u e. The dec ease
ends o each a pla eau alue a high empe a u e. In some
cases he E′p ofile e en ually e ol es o an inc ease in alue,
indica ing a ce ain he mose ing po en ial. DMA p ofiles o he
addi i e- ee, u ea and sodium bisulfi e specimens do no display
any significan diffe ence, in spi e o he effec obse ed on he
isco-elas ic p ope ies o hei co esponding blends (Fig. 4A).
Sodium sulfi e-con aining specimens show lowe E′ alues a low
empe a u e bu highe alues in he high empe a u e egion
as compa ed o he e e ence (addi i e- ee) sys em. This sys em
also e ol es in a mo e g adual way han he o he bioplas ic
specimens, pa icula ly a high empe a u e, showing no he -
mose ing po en ial egion. This beha iou may be associa ed
wi h he abili y o sodium sulfi e o impai disulfide b idges. Thus,
he esul s ob ained a e applica ion o he p ocedu e de ised by
Be e idge e al.38 indica e ha he concen a ion o ee sul hyd yl
g oups in c ayfish flou is inc eased om 19 ±3 mmol kg−1 o
300 ±17 mmol kg−1a e addi ion o 3 g sodium sulfi e kg−1
p o ein.
As o L-cys eine specimens, hey show a a he simila DMA
p ofile a low and medium empe a u e bu he e is also an
appa en inc ease in E′a high empe a u e, which e eals ha his
sys em s ill exhibi s a ma ked emnan he mose ing po en ial o
u he p ocessing.
AB
Figu e 5. S o age modulus (E′) alues om dynamic mechanical analysis (DMA) empe a u e amp measu emen s ca ied ou a 1 Hz and 3 ∘Cmin
−1
o diffe en c ayfish flou /glyce ol (CF/GL) sys ems: (A) addi i e-con aining (sodium bisulfi e, sodium sulfi e, u ea o L-cys eine) specimens; and (B)
L-cys eine-con aining specimens p ocessed a diffe en moulding empe a u e (100 o 130 ∘C) and packing ime (200 o 600 s). The addi i e- ee CF/GL is
included as a e e ence. BS, sodium bisulfi e; SS, sodium sulfi e; U, u ea; LC, L-cys eine.
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C ayfish bio-based plas ic ma e ials p ocessed by injec ion moulding www.soci.o g
This inc ease may be a consequence o diffe en c oss-linking
eac ions in ol ing S—S bond o ma ion, SH–SS in e change44
and also non-disulfide bonds. Thus, Rombou s e al.45 epo ed
a hea -induced educ ion in 𝜖-amino g oups, being mos likely
he esul o isopep ide bond o ma ion in combina ion wi h
Mailla d and/o o he hea -induced eac ions. These au ho s also
ound ha lysine- and glu amine-con aining pep ides om high
molecula weigh glu enin sub-uni s also induced he o ma ion
o isopep ide bonds, he eby demons a ing ha c oss-linking
did no solely depend on he a ailabili y o cys eine o cys ine
esidues. In his sense, i is in e es ing o no e ha c ayfish p o ein
is ich in he amino acids glu amine and lysine.46
In o de o explo e his po en ial L-cys eine-con aining speci-
mens we e p ocessed using diffe en moulding condi ions o e
he packing s age, inc easing ei he he packing ime in he
mould a 100 ∘C and 20 MPa (up o 600 s) o he moulding em-
pe a u e a 20 MPa o e 200 s (up o 130 ∘C). DMA esul s o
hese new L-cys eine-con aining specimens a e shown in Fig. 5B.
As may be obse ed, he inc ease in moulding ime does no
lead o any no iceable change in he DMA p ofile, which sug-
ges s ha he ime selec ed o he o me specimens is long
enough o comple e he c oss-linking s age. On he o he hand,
an inc ease in moulding empe a u e leads o a pla eau alue
o E′in he high- empe a u e egion. Howe e , his change
in moulding condi ions does no d i e any pa icula enhance-
men in he isco-elas ic bending p ope ies below 60 ∘C. This
beha iou is somehow unexpec ed since he occu ence o he
abo e-men ioned he mose ing po en ial ypically in ol es an
enhancemen o mechanical p ope ies.21,47,48 A possible explana-
ion o his lack o enhancemen may be ound in he ac ha
some deg ada ion o specimens has been obse ed a 130 ∘Cand
abo e. P obably, his deg ada ion is ela ed o o he componen s
(e.g. lipids con en ) a he han p o ein bu i may p oduce some
al e a ions o mechanical p ope ies. Thus, using a de a ed c ay-
fish p o ein concen a e ins ead o c ayfish flou would be a be e
choice in o de o p ocess a highe empe a u es.
Measu emen s o he uniaxial ensile s eng h
Figu e 6A displays he esul s o s ess–s ain cu es ob ained
om ensile s eng h measu emen s o addi i e- ee and
addi i e-con aining specimens a an addi i e/CF a io. All he
cu es exhibi a simila beha iou which consis o an ini ial linea
elas ic beha iou o high cons an s ess–s ain slope yielding
high alues o he Young’s modulus (E), ollowed by a de o ma-
ion s age wi h a con inuous dec ease in he s ess–s ain slope.
A second cons an slope is eached a he end o he plas ic de o -
ma ion s age. All he cu es e en ually each a maximum alue
o he s ess (𝜎max) and he s ain a b eak (𝜖max). Only L-cys eine
leads o an appa en enhancemen o he ensile s eng h p o-
file, also leading o a sligh ly sho e s ain alue. The alues
o hese pa ame e s (E,𝜎max and 𝜖max) and hei co esponding
s anda d de ia ions a e plo ed in Fig. 6B o he addi i e- ee and
addi i e-con aining specimens a 3 g kg−1addi i e/CF a io. This
figu e indica es once again ha addi i e L-cys eine is he only
one ha imp o es pa ame e s Eand 𝜎max o e he addi i e- ee
sys em, unde he same p ocessing condi ions. On he o he hand
L-cys eine-added specimens exhibi lowe alues o 𝜖max.The
emainde o he addi i es lead o simila o e en lowe alues
o he h ee pa ame e s. Figu e 6B also shows he alues o
L-cys eine-added specimens moulded a longe ime o highe
empe a u e. As may be obse ed, an inc ease in he packing ime
om 200 s up o 600 s does no yield any no iceable change in en-
sile pa ame e s. This esul indica es ha a e 200 s packing ime
he sp ue is al eady closed by he solidified blend. On he o he
hand, an inc ease in he mould empe a u e leads o ema kable
changes in ensile pa ame e s. Thus, he maximum s ess and,
abo e all, he s ain a b eak unde go an appa en inc ease in
alue (app ox. 12% and 70%, espec i ely), whe eas he Young’s
modulus clea ly dec eases (app ox. 30%) by inc easing he mould
empe a u e om 100 o 130 ∘C. In e es ingly, he maximum
elonga ion is he p ope y ha unde goes he mos ema kable
enhancemen by a ou ing hea -induced c oss-linking. In his
way, he ma e ial exhibi s g ea e oughness, in spi e o being
less s ong. In ac , as s a ed by Lag ain e al.,49 inc easing he
elonga ion a b eak o glassy, amo phous polyme s ypically goes
a he expense o he elas ic modulus. This compensa ion may
also affec he bending elas ic p ope ies o he bioplas ic, hus
explaining he small dependence o DMA p ofiles on moulding
empe a u e.
Mo eo e , his beha iou is simila o ha ound o o he elas-
ome ic ma e ials such as ubbe -based blends50 and is consis en
wi h he esul s om DMA measu emen s ha show an ex ension
o he ubbe y pla eau ob ained a high empe a u e.
AB
Figu e 6. Resul s om ensile s eng h measu emen s o diffe en c ayfish flou /glyce ol (CF/GL) sys ems: addi i e- ee o 3 g kg−1addi i e-con aining
specimens (sodium bisulfi e, sodium sulfi e, u ea o L-cys eine). (A) S ess--s ain cu es, and (B) pa ame e s om ensile s eng h measu emen s:
maximum s ess (𝜎max), s ain a b eak (𝜖max) and Young’s modulus (E). BS, sodium bisulfi e; SS, sodium sulfi e; U, u ea; LC, L-cys eine.
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686
www.soci.o g M Felix e al.
CONCLUSIONS
F om he expe imen al esul s, i may be concluded ha moni o -
ing he o que o e mixing o p o ein-based flou , addi i es and
plas icise i is use ul o selec he mo e sui able condi ions (e.g.
mixing ime and o mula ion) in e ms o ene gy efficiency. Cha ac-
e isa ion o he heological p ope ies o blends (pa icula ly hei
dependence on empe a u e) is also impo an o selec sui able
ope a ion condi ions o injec ion moulding p ocessing.
The addi ion o educing agen (sodium bisulfi e o sodium
sulfi e), dena u ing agen (u ea) o c oss-linking p omo e
(L-cys eine), always yields an inc ease in ene gy efficiency (i.e. a
dec ease in REI alue) a he mixing s age, leading o a ema kable
educ ion in he linea isco-elas ic p ope ies o blends.
In con as , he effec o he addi i es on he mechanical p op-
e ies o he final bioplas ic ma e ial is no so clea , pa icula ly o
he esul s om DMA measu emen s. The addi i e de eloping a
g ea e effec is L-cys eine, which p o ides specimens showing a
highe alue o he Young’s modulus, as well as a emnan he -
mose ing po en ial o u he p ocessing a high empe a u e.
This po en ial ypically in ol es an enhancemen o mechanical
p ope ies.21,47,48,51 Howe e , in his s udy he maximum elonga-
ion is he p ope y ha is ema kably enhanced by inc easing he
he mose ing empe a u e. This moulding empe a u e-d i en
enhancemen is clea ly a he expense o he Young’s modulus,
such ha he effec on he bending elas ic modulus is dampened.
The p esen wo k pu s o wa d he easibili y o de eloping
c ayfish-based g een biodeg adable plas ics, he eby finding
po en ial alue-added applica ions o his p o ein concen a e
by-p oduc . Howe e , much esea ch is s ill needed in o de o
u he explo e he po en ial o c ayfish in his field. Thus, om he
esul s ob ained, i seems o be an ad isable s a egy o in es iga e
he possible syne ge ic effec s esul ing om he combina ion o
he diffe en addi i es s udied. This combina ion could be s udied
by inco po a ing each addi i e a he mixing s age, acco ding o
he ollowing sequence: (1) u ea, o acili a e dis up ion o physical
in e ac ions; (2) sodium sulfi e, o induce b eakage o disulfide
bonds; and (3) L-cys eine o an icipa e and imp o e mixing effi-
ciency and o p omo e subsequen c oss-linking a he moulding
s age.
ACKNOWLEDGEMENTS
This wo k is pa o a esea ch p ojec sponso ed by Andalu-
sian Go e nmen (Spain) (p ojec TEP-6134) and by he ‘Minis-
e io de Economía y Compe i i idad’ om Spanish Go e nmen
(Re . MAT2011-29275-C02-02). The au ho s g a e ully acknowl-
edge hei financial suppo . The au ho s also acknowledge he
Mic oanalysis Se ice (CITIUS-Uni e sidad de Se illa) o p o iding
ull access and assis ance wi h he LECO-CHNS-932 equipmen .
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