Ci a ion: Mok ejš, P.; Gál, R.;
Pa laˇcko á, J. Enzyme Condi ioning
o Chicken Collagen and Taguchi
Design o Expe imen s Enhancing he
Yield and Quali y o P epa ed
Gela ins. In . J. Mol. Sci. 2023,24,
3654. h ps://doi.o g/10.3390/
ijms24043654
Academic Edi o : And eas Taube
Recei ed: 27 Janua y 2023
Accep ed: 9 Feb ua y 2023
Published: 11 Feb ua y 2023
Copy igh : © 2023 by he au ho s.
Licensee MDPI, Basel, Swi ze land.
This a icle is an open access a icle
dis ibu ed unde he e ms and
condi ions o he C ea i e Commons
A ibu ion (CC BY) license (h ps://
c ea i ecommons.o g/licenses/by/
4.0/).
In e na ional Jou nal o
Molecula Sciences
A icle
Enzyme Condi ioning o Chicken Collagen and Taguchi Design
o Expe imen s Enhancing he Yield and Quali y o
P epa ed Gela ins
Pa el Mok ejš 1,* , Robe Gál2and Jana Pa laˇcko á3
1Depa men o Polyme Enginee ing, Facul y o Technology, Tomas Ba a Uni e si y in Zlín, Va eˇcko a 275,
760 01 Zlín, Czech Republic
2Depa men o Food Technology, Facul y o Technology, Tomas Ba a Uni e si y in Zlín, Va eˇcko a 275,
760 01 Zlín, Czech Republic
3
Depa men o Lipids, De e gen s and Cosme ics Technology, Facul y o Technology, Tomas Ba a Uni e si y in
Zlín, Va eˇcko a 275, 760 01 Zlín, Czech Republic
*Co espondence: mok [email p o ec ed]; Tel.: +42-05-7603-1230
Abs ac :
Du ing he p oduc ion o mechanically deboned chicken mea (MDCM), a by-p oduc is
c ea ed ha has no adequa e use and is mos ly disposed o in ende ing plan s. Due o he high con en
o collagen, i is a sui able aw ma e ial o he p oduc ion o gela in and hyd olysa es. The pu pose
o he pape was o p ocess he MDCM by-p oduc in o gela in by 3-s ep ex ac ion. An inno a i e
me hod was used o p epa e he s a ing aw ma e ial o gela in ex ac ion, demine aliza ion in HCl,
and condi ioning wi h a p o eoly ic enzyme. A Taguchi design wi h wo p ocess ac o s (ex ac ion
empe a u e and ex ac ion ime) was used a h ee le els (42, 46, and 50
◦
C; 20, 40, and 60 min)
o op imize he p ocessing o he MDCM by-p oduc in o gela ins. The gel- o ming and su ace
p ope ies o he p epa ed gela ins we e analyzed in de ail. Depending on he p ocessing condi ions,
gela ins a e p epa ed wi h a gel s eng h o up o 390 Bloom, a iscosi y o 0.9–6.8 mPa
·
s, a mel ing
poin o 29.9–38.4
◦
C, a gelling poin o 14.9–17.6
◦
C, excellen wa e - and a -holding capaci y,
and good oaming and emulsi ying capaci y and s abili y. The ad an age o MDCM by-p oduc
p ocessing echnology is a e y high deg ee o con e sion (up o 77%) o he s a ing collagen aw
ma e ial o gela ins and he p epa a ion o 3 quali a i ely di e en gela in ac ions sui able o a
wide ange o ood, pha maceu ical, and cosme ic applica ions. Gela ins p epa ed om MDCM
by-p oduc can expand he o e o gela ins om o he han bee and po k issues.
Keywo ds:
bioma e ials; by-p oduc ; enzyme condi ioning; collagen; gela in; mechanically deboned
chicken mea ; Taguchi design; ze o-was e
1. In oduc ion
Gela in is one o he mos e sa ile biopolyme s, and due o i s unique ilm, gel, and
su ace p ope ies, i is widely used in he ood, pha macy, cosme ics, and pho og aphy
indus ies, as well as in he p oduc ion o packaging ma e ials and encapsula es and in
a numbe o echnical applica ions [
1
–
3
]. This is e idenced by he global p oduc ion o
gela in, which ep esen ed app oxima ely 700 kilo ons in 2021; he o al u no e in e ms
o aw ma e ial ep esen s app oxima ely 3500 million USD. O his amoun , app oxima ely
30% was consumed in he p oduc ion o ood and be e ages, 25% in nu aceu icals, 19%
in pha maceu icals, 14% in pho og aphy, 7% in pe sonal ca e p oduc s, and 5% in o he
applica ions. A u he inc ease in gela in p oduc ion is expec ed o 2025 by app oxima ely
6.0% compa ed o 2019 [
4
]. Gela in can be made om any animal issue ha con ains
collagen. Cu en ly, app oxima ely 95% o all gela in is p oduced indus ially om bee
and po k issues. The es consis s o al e na i e sou ces o collagen which ha e gained
impo ance in he las 20 yea s no only due o he g owing demand o gela in bu also
In . J. Mol. Sci. 2023,24, 3654. h ps://doi.o g/10.3390/ijms24043654 h ps://www.mdpi.com/jou nal/ijms
In . J. Mol. Sci. 2023,24, 3654 2 o 22
due o special consume equi emen s [
5
]. Some examples a e eligious o cul u al easons
o ejec ing po k o bee p oduc s o consume p e e ences o ish o poul y p oduc s
o e bee and po k. I is also necessa y o men ion he socially changing a i udes owa ds
handling animal by-p oduc s and he possibili ies o hei use (philosophy o he ci cula
economy). Gela ins can be p epa ed om a ious unused pa s o poul y, mos commonly
chicken ee and skin [
6
,
7
], duck ee and skin [
8
,
9
], and chicken bones [
10
,
11
]; o he ypes
o poul y a e less common [
12
]. F om ish (bo h eshwa e and ma ine), gela ins a e mos
o en p epa ed om skin, bones, scales, ins, o heads [
13
–
17
]. The condi ions o p epa ing
gela in om og skin a e also known [
18
]. Howe e , he disad an age o al e na i e aw
ma e ial sou ces con aining collagen is hei non-s anda d pa ame e s, which signi ican ly
complica es hei p ocessing in o gela ins wi h p ope ies sui able o speci ic applica ions.
Fo example, gela ins p epa ed om cold-wa e ish species a e less s able and ha e wo se
heological p ope ies, which also complica es hei p ocessing. The e a e also undamen al
di e ences in he p ope ies o gel o ma ion (gel s eng h, gelling, and mel ing poin )
be ween gela ins p epa ed om cold and wa m wa e ish [19–22].
When p ocessing collagen aw ma e ials (mainly skin and endons) in o gela ins, i
is necessa y o emo e accompanying componen s (mos o en a , globula p o eins, and
glycop o eins) om he s a ing aw ma e ial and o p epa e he aw ma e ial in a sui able
way o con olled ex ac ion. Fo his pu pose, adi ional o al e na i e p ocedu es a e
used—namely condi ioning in an acidic o alkaline en i onmen and a ely he use o
enzymes [
2
,
23
]. The excep ion is o bones o which demine aliza ion is necessa y. This
is done in an acidic en i onmen [
24
]. Gela in ex ac ion is ca ied ou wi h ho wa e
(depending on he ype o aw ma e ial a a empe a u e o 40
◦
C minimum) in ex ac o s o
a ious designs. In he indus ial p oduc ion o bee and po k gela in, mul is age ex ac ion
is used o e icien ly con e collagen in o gela in [2].
Mechanically deboned mea can be ob ained om all animals, wi h he excep ion
o uminan s, which ha e been banned as a aw ma e ial since 2011 due o conce ns
abou he possible disease o bo ine spongi o m encephalopa hy (BSE). Mechanically
deboned chicken mea (MDCM) is ob ained mos o en and used o he p oduc ion o
mea p oduc s [
25
,
26
]. I is ob ained by mechanical sepa a ion o he emaining pa s o
he mea , which a e ound in he bones and ibs a e he mea has been cu and can make
up o 30% o he muscle con en . To ob ain MDCM, a adi ional sepa a ion p ocedu e is
used, which is based on p essing bone aw ma e ials; a con inuous illing and p essing
me hod o a sepa a e illing and p essing p ocess can be applied. Du ing he p essing
echnique, he muscle wi h he a y and connec i e pa s is sepa a ed om he bones and
ough connec i e issues. When he MDCM sepa a ion decan a ion p ocedu e is applied,
bone aw ma e ials a e g ound wi h he addi ion o lake ice and a sodium ni i e cu ing
sal mix u e. The esul ing liquid homogena e is con inuously cen i uged based on he
p inciple o decan a ion and immedia ely ozen. Sc ew con eyo s, hyd aulic pis ons, o
d um sepa a o s a e used o sepa a ion; he yield and quali y o MDCM can be egula ed,
o example, by he size o he holes in he sepa a ion sie es o he low a e o c ushed
mea and bone aw ma e ial [
27
]. In he MDCM sepa a ion p ocess, highe p essu es
a e some imes used o inc ease he yield, esul ing in a highe Ca con en in he MDCM.
Due o he p esence o a highe amoun o mine al subs ances, MDCM has good wa e
binding capaci y and is sui able as an addi ion o sausages, pâ és o poul y semi-p oduc s;
addi ions up o 10% do no nega i ely a ec he p ope ies o he inal p oduc s [
28
,
29
].
MDCM has a limi ed shel li e, which is ela ed o he possibili y o mic obial con amina ion,
he inc ease in empe a u e du ing he sepa a ion p ocess, and he highe pH alue due
o he Ca
3
(PO
4
)
2
con en . The solid esidue a e MDCM p oduc ion is cha ac e ized by
a high con en o p o eins (up o 40% in d y ma e ), a s (25–30% in d y ma e ), and
mine als (app oxima ely 30% in d y ma e ) and hus ep esen s an impo an sou ce o
aw ma e ials ich in nu ien s.
In addi ion o he basic physicochemical p ope ies o gela in (composi ion, swelling,
solubili y, colo , cla i y, odo , and as e), he main a ibu es ha bes de ine he comme cial
In . J. Mol. Sci. 2023,24, 3654 3 o 22
quali y o gela in include gel s eng h and iscosi y [
30
]. Howe e , he complex quali y o
gela ins is de e mined by a se o gel- o ming and su ace p ope ies. These a e impo an
no only o he applica ion o gela in in inal p oduc s bu also o he choice o a sui able
p ocessing echnology (ex usion, cas ing, dipping, injec ion). The gel- o ming p ope ies
also include he gelling poin (GP), mel ing poin (MP), wa e holding capaci y (WHC), and
a binding capaci y (FBC). Su ace p ope ies include oaming capaci y (FC) and oaming
s abili y (FS), emulsi ying capaci y (EC), emulsion s abili y (ES), ilm- o ming abili y, and
adhesi e and cohesi e p ope ies. The p ope ies o gela ins depend on many ac o s,
especially he ype o collagen (bee , po k, ish, poul y), he condi ions o collagen p ocess-
ing (acidic, alkaline, enzyme, combined), he condi ions o gela in ex ac ion (especially
empe a u e, pH, ime), and he me hods o p ocessing he ex ac ed gela in (especially he
choice o d ying me hod) [
31
]. The ype o collagen and he p ocessing condi ions a ec
he amino acid composi ion o gela in and he dis ibu ion o molecula weigh s [
32
,
33
].
The ep esen a ion and a io be ween
α
-,
β
-, and
γ
-chains in gela in a ec s he iscosi y
o gela in ( iscosi y inc eases as he amoun o
β
-
γ
-chains inc eases) [
34
]; i also a ec s
he GP and MP o gela in (a highe ep esen a ion o
α
-chains shi s bo h empe a u es
o highe alues) [
31
]. The s uc u al s abili y o gela in is mainly due o he con en o
he amino acids p oline and hyd oxyp oline, which con ibu e o he s abiliza ion o he
s uc u e by means o hyd ogen b idges [
35
]. A highe con en o hese amino acids will
be e lec ed in an inc ease in he GP and MP o gela in [
36
]. Mo e de ailed in o ma ion
on he s uc u e o gela in is p o ided by heological measu emen s [
37
], scanning and
ansmission elec on mic oscopy (SEM and TEM) [
38
], Fou ie ans o m in a ed (FTIR)
spec oscopy [39], and di e en ial scanning calo ime y (DSC) [40].
In ou p e ious s udy de o ed o he p epa a ion o gela in om he MDCM by-p oduc ,
a wo-le el ac o ial expe imen wi h h ee s udied p ocess ac o s was used [
41
]. Compa ed
o s udies de o ed o he p epa a ion o gela in om he same aw
ma e ial [42–44]
, in
ou wo k highe gela in yields we e achie ed. In ou s udy, he basic p ope ies o gela in
(gel s eng h, iscosi y, ash con en ) we e de e mined. I is clea ha all s udies showed
impo an esul s in ega d o he p ocessing o p e iously unused MDCM by-p oduc s
in o gela ins. Conside ing he g ea po en ial o his aw ma e ial sou ce, i would be
ad isable o deal wi h a mo e de ailed op imiza ion o he gela in p epa a ion p ocedu e,
a ho ough cha ac e iza ion o gela in, and he p oposal o i s applica ions wi h ega d o
hei p ope ies.
The objec i es o he cu en s udy a e as ollows: (1) Op imize he p ocess o p epa ing
gela in om he MDCM by-p oduc o achie e he maximum deg ee o con e sion o he
s a ing aw ma e ial o gela ins wi hou a nega i e e ec on hei quali y. Fo his pu pose,
we p opose an inno a i e p ocess and Taguchi design o expe imen s: demine aliza ion o
he MDCM by-p oduc , enzyme condi ioning o he pu i ied collagen, and 3-s age gela in
ex ac ion; (2) design he p ocessing echnology so as o limi he numbe o by-p oduc s
c ea ed; (3) pe o m a comp ehensi e assessmen o he quali y o p epa ed gela ins by
de e mining hei gel- o ming and su ace p ope ies; (4) p opose po en ial indus ial
applica ions o he p epa ed gela ins. Scien i ic hypo heses: By adjus ing he p ocess
condi ions du ing he p ocessing o collagen om MDCM by-p oduc in o gela ins, gela ins
a e p epa ed wi h a highe yield han using s anda d echnological p ocedu es. The highe
yields o gela in will no ha e a nega i e e ec on hei p ope ies.
2. Resul s
The esul s o p ocessing he MDCM by-p oduc in o h ee ac ions o gela ins a e
p esen ed in he ollowing ou subsec ions.
2.1. Mass Balance o he P ocess
The schedule o expe imen s and esul s o he p ocessing o he MDCM by-p oduc
in o h ee gela in ac ions a e p esen ed in Table 1. Table 2shows he esul s o he analysis
o a iance o he gela in yields.
In . J. Mol. Sci. 2023,24, 3654 4 o 22
Table 1. The expe imen al design and he esul s o he p ocess mass balance.
Exp.
No.
Fac o
A (◦C)
Fac o B
(min)
YH
(%)
YG1
(%)
YG2
(%)
YG3
(%)
UR
(%)
MBE
(%)
YG∑
(%)
1 42 20 10.6 6.2 39.7 8.0 32.6 2.9 53.9
2 42 40 12.0 22.5 44.8 3.2 14.2 3.3 70.5
3 42 60 11.1 25.4 49.3 2.1 8.9 3.2 76.8
4 46 20 12.4 45.4 24.2 2.9 11.8 3.3 72.5
5 46 40 11.7 38.4 30.4 4.0 12.2 3.3 72.8
6 46 60 11.0 19.2 49.8 3.9 14.6 1.5 72.9
7 50 20 12.1 29.1 30.8 7.2 17.8 3.0 67.1
8 50 40 11.6 30.5 27.1 7.3 21.1 2.4 64.9
9 50 60 10.8 32.8 22.4 7.2 21.9 4.9 62.4
10 * 46 40 3.3 1.3 2.4 4.4 86.9 1.7 8.1
Fac o A—ex ac ion empe a u e a 1s ex ac ion s ep; Fac o B—ex ac ion ime a 1s ex ac ion s ep; Y
H
— he
yield o collagen hyd olysa e; Y
G1
— he yield o he 1s gela in ac ion; Y
G2
— he yield o he 2nd gela in ac ion;
Y
G3
— he yield o he 3 d gela in ac ion; UR—an undissol ed esidue; MBE— he mass balance e o ; Y
G∑
— o al
gela in ex ac ion yield; * Exp. No. 10—a blind expe imen (no enzyme condi ioning).
Table 2. Analysis o a iance o he expe imen al design o gela in yields.
Deg ee o
F eedom
Sum o
Squa es
Mean
Squa es F-Value p-Value
Response: The yield o he 1s gela in ac ion, YG1 (%) = −44.6 + 1.60A −0.028B
Reg ession 2 246.30 123.148 0.94 0.441
Fac o A
(Ex ac ion
empe a u e)
1 244.48 244.482 1.87 0.220
Fac o B
(Ex ac ion
ime)
1 1.82 1.815 0.01 0.910
E o 6 784.12 130.686
To al 8 1030.42
Response: The yield o he 2nd gela in ac ion, YG2 (%) = 129.0 −2.229A + 0.223B
Reg ession 2 596.7 298.37 5.59 0.043
Fac o A
(Ex ac ion
empe a u e)
1 477.0 477.04 8.94 0.024 •
Fac o B
(Ex ac ion
ime)
1 119.7 119.71 2.24 0.185
E o 6 320.2 53.36
To al 8 916.9
Response: The yield o he 3 d gela in ac ion, YG3 (%) = −9.4 + 0.350A −0.0408B
Reg ession 2 15.762 7.881 1.79 0.246
Fac o A
(Ex ac ion
empe a u e)
1 11.760 11.760 2.67 0.153
Fac o B
(Ex ac ion
ime)
1 4.002 4.002 0.91 0.377
E o 6 26.407 4.401
To al 8 42.169
•s a is ically signi ican ac o (p- alue ≤0.05).
Figu e 1shows he ela ionship be ween a esponse a iable (gela in yields) and
wo p edic o a iables (ex ac ion empe a u e and ex ac ion ime) using con ou plo s.
Depending on he alues o bo h s udied p ocess ac o s, he yield o he i s gela in ac ion
(Y
G1
) anges om less han 10% o mo e han 40%. The highes Y
G1
yields we e achie ed
a ex ac ion empe a u es o 45–49
◦
C wi h ex ac ion ime < 35 min (see Figu e 1a);
bo h s udied p ocess ac o s we e no ound o be signi ican a he moni o ed le el o
In . J. Mol. Sci. 2023,24, 3654 5 o 22
signi icance (p- alue
≤
0.05). The second gela in ac ion (Y
G2
) is among he dominan
gela in ac ions in e ms o pe cen age ep esen a ion, wi h yields o app oxima ely 22 o
50%; gela ins om he second ac ions show he bes gel- o ming and su ace p ope ies
(see Sec ion 2.3). F om Figu e 1b, he e is an ob ious end o Y
G2
yield g ow h, especially
wi h inc easing ex ac ion ime (Fac o B). The ex ac ion ime is a s a is ically signi ican
ac o wi h a p- alue = 0.024, see Table 1. On he con a y, i is e iden om he con ou
posi ion ha he ex ac ion empe a u e (Fac o A) has a smalle e ec on Y
G2
; he p- alue
is highe han 0.05. The hi d gela in ac ions, wi h hei app oxima e yield (Y
G3
) o 2–8%,
ha e he lowes ep esen a ion o ex ac ed gela ins, see Figu e 1c. Nei he o he wo
moni o ed p ocess ac o s is s a is ically signi ican (p- alues a e > 0.05). Figu e 1d hen
shows he o al yield o ex ac ed gela in, Y
G∑
(sum o Y
G1
, Y
G2
, and Y
G3
). I is ob ious
ha a an app op ia ely chosen ex ac ion empe a u e (42–44
◦
C) and an ex ac ion ime o
50–60 min, he deg ee o collagen- o-gela in con e sion is e y high, up o app oxima ely
75%.
In . J. Mol. Sci. 2023, 24, x FOR PEER REVIEW 5 o 23
Figu e 1 shows he ela ionship be ween a esponse a iable (gela in yields) and wo
p edic o a iables (ex ac ion empe a u e and ex ac ion ime) using con ou plo s.
Depending on he alues o bo h s udied p ocess ac o s, he yield o he i s gela in ac ion
(YG1) anges om less han 10% o mo e han 40%. The highes YG1 yields we e achie ed a
ex ac ion empe a u es o 45–49 °C wi h ex ac ion ime < 35 min (see Figu e 1a); bo h
s udied p ocess ac o s we e no ound o be signi ican a he moni o ed le el o signi icance
(p- alue ≤ 0.05). The second gela in ac ion (YG2) is among he dominan gela in ac ions in
e ms o pe cen age ep esen a ion, wi h yields o app oxima ely 22 o 50%; gela ins om
he second ac ions show he bes gel- o ming and su ace p ope ies (see Sec ion 2.3). F om
Figu e 1b, he e is an ob ious end o YG2 yield g ow h, especially wi h inc easing ex ac ion
ime (Fac o B). The ex ac ion ime is a s a is ically signi ican ac o wi h a p- alue = 0.024,
see Table 1. On he con a y, i is e iden om he con ou posi ion ha he ex ac ion
empe a u e (Fac o A) has a smalle e ec on YG2; he p- alue is highe han 0.05. The hi d
gela in ac ions, wi h hei app oxima e yield (YG3) o 2–8%, ha e he lowes ep esen a ion
o ex ac ed gela ins, see Figu e 1c. Nei he o he wo moni o ed p ocess ac o s is
s a is ically signi ican (p- alues a e > 0.05). Figu e 1d hen shows he o al yield o ex ac ed
gela in, YG∑ (sum o YG1, YG2, and YG3). I is ob ious ha a an app op ia ely chosen ex ac ion
empe a u e (42–44 °C) and an ex ac ion ime o 50–60 min, he deg ee o collagen- o-
gela in con e sion is e y high, up o app oxima ely 75%.
Figu e 1. The in luence o ex ac ion empe a u e a 1s ex ac ion s ep and ex ac ion ime a 1s
ex ac ion s ep on gela in yields: (a) he yield o he 1s gela in ac ion; (b) he yield o he 2nd
gela in ac ion; (c) he yield o he 3 d gela in ac ion; (d) he o al yield o gela ins.
I we compa e he yields o gela ins (YG1, YG2, and YG3) p epa ed acco ding o ou
p oposed p ocedu e consis ing o demine aliza ion o he s a ing aw ma e ial, enzyme
condi ioning o collagen, and 3-s age gela in ex ac ion acco ding o Taguchi design (see
Exp. Nos. 1–9 in Table 1) wi h a blind expe imen unde condi ions co esponding o he
mean alues o he moni o ed ac o s (ex ac ion empe a u e 46 °C and ex ac ion ime
40 min) wi hou enzyme collagen condi ioning (see Exp. No. 10 in Table 1), i is e iden
Figu e 1.
The in luence o ex ac ion empe a u e a 1s ex ac ion s ep and ex ac ion ime a 1s
ex ac ion s ep on gela in yields: (
a
) he yield o he 1s gela in ac ion; (
b
) he yield o he 2nd gela in
ac ion; (c) he yield o he 3 d gela in ac ion; (d) he o al yield o gela ins.
I we compa e he yields o gela ins (Y
G1
, Y
G2
, and Y
G3
) p epa ed acco ding o ou
p oposed p ocedu e consis ing o demine aliza ion o he s a ing aw ma e ial, enzyme
condi ioning o collagen, and 3-s age gela in ex ac ion acco ding o Taguchi design (see
Exp. Nos. 1–9 in Table 1) wi h a blind expe imen unde condi ions co esponding o he
mean alues o he moni o ed ac o s (ex ac ion empe a u e 46
◦
C and ex ac ion ime
40 min) wi hou enzyme collagen condi ioning (see Exp. No. 10 in Table 1), i is e iden ha
he inno a i e me hod o collagen condi ioning has a undamen al e ec on gela in yield.
The o al yield o gela in (Y
G∑
) in he blind expe imen is only 8.1%, which is app oxima ely
9 imes less han ha o gela in ex ac ed unde he same p ocess condi ions (Exp. No. 5)
wi h enzyme collagen condi ioning. Compa ed wi h he yield o gela ins p epa ed unde
di e en condi ions (Exp. Nos. 1–9), he Y
G∑
in he blind expe imen is 6.7–9.5 imes lowe .

In . J. Mol. Sci. 2023,24, 3654 6 o 22
2.2. Fi s Gela in F ac ions
The esul s o he p ope ies analysis o he i s gela in ac ions p epa ed om he
MDCM by-p oduc a e shown in Table 3.
Table 3. Resul s o he analysis o he p ope ies o he i s gela in ac ions.
P ocess Fac o s Gela in P ope ies
Exp.
No.
Fac o
A (◦C)
Fac o
B (min)
Ash
(%)
υ
(mPa·s)
WHC
(%)
FBC
(%)
FC
(%)
FS
(%)
EC
(%)
ES
(%)
1 42 20 1.17 1.7 220 840 8 0 47 93
2 42 40 0.97 1.6 220 900 8 2 47 92
3 42 60 1.23 1.5 230 920 7 2 48 93
4 46 20 0.88 1.6 230 1090 6 2 48 93
5 46 40 0.96 1.5 230 1090 7 2 48 94
6 46 60 1.43 1.5 240 1110 7 2 47 93
7 50 20 1.39 1.5 240 1140 8 3 47 93
8 50 40 1.02 1.4 250 1210 8 4 48 95
9 50 60 1.16 1.4 240 1210 7 3 46 94
10 * 46 40 1.02 1.6 240 1150 7 4 48 93
Fac o A— empe a u e a 1s ex ac ion s ep; Fac o B—ex ac ion ime a 1s ex ac ion s ep;
υ
— iscosi y;
WHC—wa e holding capaci y; FBC— a binding capaci y; FC— oaming capaci y; FS— oaming s abili y; EC—
emulsi ying capaci y; ES—emulsion s abili y; * Exp. No. 10—a blind expe imen (no enzyme condi ioning).
None o he gela ins ob ained in he i s ex ac ion s ep o med measu able gels;
he e o e, hese a e ze o Bloom alue gela ins. The ze o Bloom alue is also ela ed o
he iscosi y o gela in, which eaches e y low alues (1.4–1.7 mPa
·
s), ega dless o he
changing ex ac ion condi ions. Simila ly, i is wi h WHC, whe e no signi ican di e ence
be ween gela ins is appa en ; depending on ex ac ion condi ions, WHC = 220–250%. Fo
FBC, a sligh g ow h end is e iden wi h inc easing ex ac ion empe a u e and, a he
same ime, p olonging ex ac ion ime; om alues sligh ly exceeding 800% a he minimum
alues o bo h moni o ed ac o s o app oxima ely 1200% a he uppe limi s o he ac o s.
Foaming p ope ies, FC and FS, a e e y low, 6 o 8% o 0 o 4%, espec i ely; empe a u e
and ex ac ion ime do no undamen ally a ec hese pa ame e s. I is simila o he
emulsi ying p ope ies, EC and ES, o which p ocess condi ions do no a ec hei changes.
Howe e , all gela ins ha e e y good EC alues (46–48%) and excellen ES (92–95%). The
p ope ies o gela in p epa ed unde he condi ions o a blind expe imen (wi hou enzyme
condi ioning) unde condi ions co esponding o he mean alues o he moni o ed ac o s
(ex ac ion empe a u e 46
◦
C and ex ac ion ime 40 min)–see Exp. No. 10 in Table 3–do
no undamen ally di e om he p ope ies o gela in p epa ed in Exp. Nos. 1–9.
2.3. Second Gela in F ac ions
The esul s o he p ope ies analysis o he second gela in ac ions p epa ed om
he MDCM by-p oduc a e shown in Table 4. Table 5shows he esul s o he analysis o
a iance o he s eng h o he gela in gel, he iscosi y, he mel ing poin , and he gelling
poin .
The ash con en is e y low in all gela ins p epa ed acco ding o he Taguchi design
(Exp. Nos. 1–9); i a ies be ween 0.34–0.70%. Fundamen al di e ences we e no ound
in he wa e holding capaci y (930–1090%) and a binding capaci y (980–1470%). Gela in
p epa ed acco ding o he condi ions o Exp. No. 9 has a signi ican ly highe oaming
capaci y (36%) han he o he gela ins (18–22%); he e is a simila di e ence in oaming
s abili y (24% e sus 8–18%). In e ms o emulsi ying capaci y and emulsion s abili y, he e
a e no undamen al di e ences be ween he gela ins p epa ed acco ding o expe imen s
1–9.
In . J. Mol. Sci. 2023,24, 3654 7 o 22
Table 4. Resul s o he analysis o he p ope ies o he second gela in ac ions.
P ocess Fac o s Gela in P ope ies
Exp.
No.
Fac o
A
(◦C)
Fac o
B
(min)
GS
(Bloom)
MP
(◦C)
GP
(◦C)
υ
(mPa·s)
Ash
(%)
WHC
(%)
FBC
(%) FC (%) FS (%) EC (%) ES (%)
1 42 20 174 35.3 16.6 2.2 0.66 1010 1310 20 16 48 93
2 42 40 80 28.9 15.0 1.6 0.35 930 980 18 8 46 93
3 42 60 125 32.3 15.3 1.8 0.34 970 1390 18 10 47 93
4 46 20 143 32.8 15.5 1.9 0.43 950 1070 22 18 48 93
5 46 40 105 30.4 14.9 2.0 0.36 960 1170 20 8 51 90
6 46 60 262 36.8 17.1 2.9 0.45 960 1230 20 16 48 93
7 50 20 284 37.9 16.7 2.7 0.46 980 1250 20 16 47 93
8 50 40 269 35.1 16.4 2.6 0.43 990 1470 20 18 49 90
9 50 60 290 38.4 17.6 3.8 0.70 1090 1460 36 24 52 92
10 * 46 40 460 35.1 26.8 6.8 0.54 1320 1540 40 28 54 93
Fac o A— empe a u e a 1s ex ac ion s ep; Fac o B—ex ac ion ime a 1s ex ac ion s ep; GS—gel s eng h;
MP—mel ing poin ; GP—gelling poin ;
υ
— iscosi y; WHC—wa e holding capaci y; FBC— a binding capaci y;
FC— oaming capaci y; FS— oaming s abili y; EC—emulsi ying capaci y; ES—emulsion s abili y; * Exp. No. 10—a
blind expe imen (no enzyme condi ioning).
Table 5.
Analysis o a iance o he expe imen al design o gela in gel s eng h, gela in iscosi y,
mel ing poin , and gelling poin .
Deg ee o
F eedom
Sum o
Squa es Mean Squa es F- alue p-Value
Response: Gel s eng h (Bloom) = −723 + 19.33A + 0.64B
Reg ession 2 36,870.8 18,435.4 5.69 0.041
Fac o A
(Ex ac ion
empe a u e)
1 35,882.7 35,882.7 11.07 0.016 •
Fac o B
(Ex ac ion
ime)
1 988.2 988.2 0.30 0.601
E o 6 19,451.2 3241.9
To al 8 56,322.0
Response: Viscosi y (mPa·s) = −4.89 + 0.1458A + 0.01417B
Reg ession 2 2.5233 1.2617 5.98 0.037
Fac o A
(Ex ac ion
empe a u e)
1 2.0417 2.0417 9.68 0.021 •
Fac o B
(Ex ac ion
ime)
1 0.4817 0.4817 2.28 0.182
E o 6 1.2656 0.2109
To al 8 3.7889
Response: Me ing poin (◦C) = 5.2 + 0.621A + 0.0125B
Reg ession 2 37.3767 18.6883 2.21 0.191
Fac o A
(Ex ac ion
empe a u e)
1 37.0017 37.0017 4.37 0.082
Fac o B
(Ex ac ion
ime)
1 0.3750 0.3750 0.04 0.840
E o 6 50.8322 8.4720
To al 8 88.2089
Response: Gelling poin (◦C) = 8.44 + 0.1583A + 0.0100B
Reg ession 2 2.6467 1.3233 1.60 0.277
Fac o A
(Ex ac ion
empe a u e)
1 2.4067 2.4067 2.92 0.138
Fac o B
(Ex ac ion
ime)
1 0.2400 0.2400 0.29 0.609
E o 6 4.9489 0.8248
To al 8 7.5956
•s a is ically signi ican ac o (p- alue ≤0.05).
In . J. Mol. Sci. 2023,24, 3654 8 o 22
P ope ies o gela in p epa ed unde he condi ions o a blind expe imen (wi hou
enzyme condi ioning) unde condi ions co esponding o he mean alues o he moni o ed
ac o s (ex ac ion empe a u e 46
◦
C and ex ac ion ime 40 min)–see Exp. No. 10 in Table 4–
di e s signi ican ly in some pa ame e s om he p ope ies o he gela ins p epa ed in Exp.
Nos. 1–9. In pa icula , his is a e y high gel s eng h alue, which is 1.6 o 5.8 imes highe
compa ed o gela ins p epa ed om Exp. Nos. 1–9; o iscosi y, he alue is
1.8–4.3 imes
highe . WHC (1320% e sus 930–1090%) and FBC (1540% e sus 980–1470%) a e also
highe . This is also ue o FC (40% e sus 18–36%) and FS (28% e sus 8–24%). The e a e
no undamen al di e ences in EC and ES o gela in om Exp. No. 10 compa ed o gela ins
p epa ed acco ding o Exp. Nos. 1–9.
Figu e 2shows he ela ionship be ween he esponse a iables and wo p edic o
a iables (ex ac ion empe a u e and ex ac ion ime) by con ou plo s. F om Figu e 2a, he
end o inc ease in gel s eng h is e iden , especially wi h inc easing ex ac ion empe a u e;
ex ac ion empe a u e is a s a is ically signi ican ac o (p- alue o 0.016; see Table 5).
Lowe gel s eng h alues (up o 200 Bloom) a e achie ed a empe a u es < 48
◦
C and
ex ac ion imes up o 50 min. Ve y good gel s eng h alues (200–250 Bloom) a e achie ed
a ex ac ion empe a u es close o he uppe limi o he obse ed empe a u e (50
◦
C),
while he ex ac ion ime does no ha e a signi ican e ec on he gel s eng h alue. A
e y simila end o in luence o ex ac ion empe a u e and ex ac ion ime on gela in
iscosi y can be seen in Figu e 2b. Gela ins wi h a lowe iscosi y (2.0–2.5 mPa
·
s) a e
p epa ed a an ex ac ion empe a u e <42.5
◦
C ega dless o he ex ac ion ime; inc easing
he ex ac ion empe a u e o 50
◦
C while simul aneously sho ening he ex ac ion ime
has he same e ec . The highes iscosi y (3.0–3.5 mPa
·
s) was achie ed a ex ac ion
empe a u es o
49–50 ◦C
wi h ex ac ion imes >55 min. The ex ac ion empe a u e is
a s a is ically signi ican ac o (p- alue = 0.021), see Table 5. An almos iden ical e ec
o bo h p ocess ac o s, as wi h gel s eng h, was eco ded on he MP; see Figu e 2c. The
mel ing poin anges om ela i ely lowe alues (a ound 30–32
◦
C) a lowe ex ac ion
empe a u es (<47
◦
C) wi hou a signi ican in luence on ex ac ion ime. A e y high MP
(35–38
◦
C) is achie ed a ex ac ion empe a u es abo e 49
◦
C; he ex ac ion ime has no
signi ican e ec on he change in MP alues. The GP is no undamen ally a ec ed by
changes in he moni o ed p ocess condi ions; i anges om 15.0 o 17.5
◦
C, wi h lowe GP
alues co esponding o lowe ex ac ion empe a u es and sho e ex ac ion ime, and
highe GP alues o ex ac ion empe a u es >49
◦
C. Bo h moni o ed p ocess ac o s a e
s a is ically insigni ican (p- alues > 0.05, see Table 5).
2.4. Thi d Gela in F ac ions
The esul s o he p ope ies analysis o he hi d gela in ac ions p epa ed om he
MDCM by-p oduc a e shown in Table 6.
F om he esul s o he hi d gela in ac ion p ope ies, gela ins p epa ed unde
Taguchi design condi ions (Exp. Nos. 1–9) can be di ided in o 3 g oups; he i s g oup
consis s o gela ins p epa ed a he lowes ex ac ion empe a u e (42
◦
C, Expe imen s 1–3),
he second gela ins p epa ed a medium ex ac ion empe a u e (46
◦
C, Expe imen s 4–6)
and he hi d gela ins p epa ed a he highes ex ac ion empe a u e (50
◦
C, Expe imen s
7–9); see Table 6. The mos undamen al is he di e ence in he s eng h o he gels.
While gela ins p epa ed a 46
◦
C did no o m gels a all and gela ins p epa ed a 42
◦
C
o med weak gels (80–88 Bloom), gela ins p epa ed a 50
◦
C had e y high gel s eng hs
(
223–230 Bloom
). The di e ences be ween MP and GP a e no undamen al be ween
gela ins wi h he abili y o o m gels. Howe e , o gela ins p epa ed a 50
◦
C, he MP
(33.9–34.8
◦
C) is highe han o gela ins p epa ed a 42
◦
C (29.2–30.8
◦
C); o GP, he e is
a di e ence be ween hese wo g oups o gela ins, 16.0–16.5
◦
C e sus 14.9–15.3
◦
C. The
g oup o gela ins p epa ed a 46
◦
C did no o m gels; he e o e, i was no possible o
de e mine MP and GP o hese gela ins. Fo iscosi y, he end is analogous o ha o gel
s eng h; he highes (2.4–2.6 mPa
·
s) in gela ins p epa ed a 50
◦
C, ollowed by gela ins
p epa ed a 42
◦
C (1.7–1.8 mPa
·
s), wi h a sligh dec ease in gela ins p epa ed a 46
◦
C.
In . J. Mol. Sci. 2023,24, 3654 9 o 22
The ash con en o all 9 p epa ed gela ins is e y low and anges om 0.48 o 0.96%. The
wa e holding capaci y is 2.6 o 3.2 imes lowe o gela ins p epa ed a 46
◦
C han o
gela ins p epa ed a 42
◦
C and e en 3.2 o 3.5 imes lowe han o gela ins p epa ed a
50
◦
C; 210–220% e sus 550 o 680% e sus 680 o 730%. The e a e no signi ican di e ences
in FBC be ween he h ee g oups o gela in; FBC = 990–1220%. On he o he hand, in FC,
gela ins p epa ed a 46
◦
C ou pe o m bo h gela ins p epa ed a 50
◦
C (18–20% e sus
16–17%) and gela ins p epa ed a 42
◦
C, which ha e a e y low FS (6–8%). Fo gela ins
p epa ed a 42 and 50
◦
C, he e is ze o FS, while o gela ins p epa ed a 46
◦
C, i is 11–12%.
The e a e no signi ican di e ences in EC and ES be ween gela ins p epa ed acco ding o
expe imen s 1–9.
In . J. Mol. Sci. 2023, 24, x FOR PEER REVIEW 9 o 23
Figu e 2. The in luence o ex ac ion empe a u e and ex ac ion ime on second gela in ac ions
p ope ies: (a) gel s eng h; (b) iscosi y; (c) mel ing poin ; (d) gelling poin .
2.4. Thi d Gela in F ac ions
The esul s o he p ope ies analysis o he hi d gela in ac ions p epa ed om he
MDCM by-p oduc a e shown in Table 6.
Table 6. Resul s o he analysis o he p ope ies o he hi d gela in ac ions.
P ocess Fac o s Gela in P ope ies
Exp.
No.
Fac o
A
(°C)
Fac o
B
(min)
GS
(Bloom)
MP
(°C)
GP
(°C)
υ
(mPa·s)
Ash
(%)
WHC
(%)
FBC
(%)
FC
(%)
FS
(%)
EC
(%)
ES
(%)
1 42 20 80 29.2 14.9 1.7 0.67 550 1190 7 0 48
95
2 42 40 82 30.1 15.0 1.
7
0.35 560 1180 6 0 47 97
3 42 60 88 30.8 15.3 1.8 0.81 680 1220 8 0 48 97
4 46 20 0 NA NA 1.4 0.96 220
1060
20 12 48 96
5 46 40 0 NA NA 1.5 0.71 210 990 18 11 47 96
6 46 60 0 NA NA 1.5 0.6
7
220 1040 20 12 48 95
7 50 20 223 33.9 16.0 2.4 0.59 680 1100 17 0 47 96
8 50 40 225 34.4 16.2 2.4 0.65 680 1120 16 0 48 96
9 50 60 230 34.8 16.5 2.6 0.48 730 1130 16 0 48 97
10* 46 40 245 34.1 15.2 2.4 0.63 910 1220 19 0 47 91
Fac o A— empe a u e a 1s ex ac ion s ep; Fac o B—ex ac ion ime a 1s ex ac ion s ep; GS—
gel s eng h; MP—mel ing poin ; GP—gelling poin ; υ— iscosi y; WHC—wa e holding capaci y;
FBC— a binding capaci y; FC— oaming capaci y; FS— oaming s abili y; EC—emulsi ying
capaci y; ES—emulsion s abili y; * Exp. No. 10—a blind expe imen (no enzyme condi ioning);
NA—no applicable.
Figu e 2.
The in luence o ex ac ion empe a u e and ex ac ion ime on second gela in ac ions
p ope ies: (a) gel s eng h; (b) iscosi y; (c) mel ing poin ; (d) gelling poin .
Table 6. Resul s o he analysis o he p ope ies o he hi d gela in ac ions.
P ocess Fac o s Gela in P ope ies
Exp.
No.
Fac o
A
(◦C)
Fac o
B
(min)
GS
(Bloom)
MP
(◦C)
GP
(◦C)
υ
(mPa·s)
Ash
(%)
WHC
(%)
FBC
(%) FC (%) FS (%) EC (%) ES (%)
1 42 20 80 29.2 14.9 1.7 0.67 550 1190 7 0 48 95
2 42 40 82 30.1 15.0 1.7 0.35 560 1180 6 0 47 97
3 42 60 88 30.8 15.3 1.8 0.81 680 1220 8 0 48 97
4 46 20 0 NA NA 1.4 0.96 220 1060 20 12 48 96
5 46 40 0 NA NA 1.5 0.71 210 990 18 11 47 96
6 46 60 0 NA NA 1.5 0.67 220 1040 20 12 48 95
7 50 20 223 33.9 16.0 2.4 0.59 680 1100 17 0 47 96
8 50 40 225 34.4 16.2 2.4 0.65 680 1120 16 0 48 96
9 50 60 230 34.8 16.5 2.6 0.48 730 1130 16 0 48 97
10* 46 40 245 34.1 15.2 2.4 0.63 910 1220 19 0 47 91
Fac o A— empe a u e a 1s ex ac ion s ep; Fac o B—ex ac ion ime a 1s ex ac ion s ep; GS—gel s eng h;
MP—mel ing poin ; GP—gelling poin ;
υ
— iscosi y; WHC—wa e holding capaci y; FBC— a binding capaci y;
FC— oaming capaci y; FS— oaming s abili y; EC—emulsi ying capaci y; ES—emulsion s abili y; * Exp. No. 10—a
blind expe imen (no enzyme condi ioning); NA—no applicable.
In . J. Mol. Sci. 2023,24, 3654 16 o 22
seconda y sou ce o p o ein in eed mix u es o a m animals and pe s, o due o i s high
p o ein con en , as a sou ce o ni ogen o he p oduc ion o plan g ow h s imula o s.
4. Ma e ials and Me hods
4.1. Ma e ials, Appliances and Chemicals
Mechanically deboned chicken mea (MDCM) by-p oduc ( om Ross 708 b oile
chicken aged 35 days) was supplied by Raciola, L d. (Uhe skýB od, Czech Republic). Fi s ,
by-p oduc ma e ial analyses we e pe o med by con en ional ood me hods [
50
–
52
]. D y
ma e con en 38.2
±
0.7%; in d y ma e : p o ein 40.3
±
1.2%, collagen (as a pa o p o ein
con en ) 79.9
±
0.5%, a 26.0
±
1.5% and ino ganic solids 29.6
±
3.8%. Each analysis was
epea ed h ee imes; mean alues and s anda d de ia ions we e calcula ed.
S e ens LFRA ex u e analyze (Leona d Fa nell and Co L d., Li e pool, UK), Ubbelo-
hde iscome e (Technisklo L d., D žko , Czech Republic), Ned o m LT 43 shake (Valašské
Meziˇ ící, Czech Republic), elec onic scale Ke n 440-47, elec onic analy ical balance Ke n
770 (Balingen, Ge many), analy ical mill IKA A 10 labo echnik (S au en, Ge many), Mem-
me ULP 400 d ying o en (B˝uchenbach, Ge many), Samsung idge eeze (Seoul, Re-
public o Ko ea), Henkelman Boxe 42 acuum packaging machine (CK ‘s-He ogenbosch,
Ne he lands), IKA T 25 digi al Ul a-Tu ax (IKA-We ke, Ge many), Wha man no. 1 pape
(Sigma Ald ich, Gillingham, UK), WTW Mul ical pH 526 pH me e (Weilheim, Ge many),
hea ing boa d Scho Ge ä e (Mainz, Ge many), a 1 mm po es size me al il e sie e (Labo -
komple , P aha, Czech Republic), o dina y labo a o y glass.
Chemicals: NaCl, NaOH, HCl, pe oleum e he , e hanol (Ve kon, P ague, Czech
Republic); all chemicals we e analy ical g ade. P o amex
®
, No ozymes endopep idase
(Copenhagen, Denma k), used o condi ioning pu i ied collagen. I is a Bacillus p o ease
complex wi h decla ed ac i i y o 1.5 AU/g; op imal wo king condi ions a e a pH 5.5 o
7.5 and empe a u e 60 ◦C. The enzyme complies wi h he ecommended pu i y speci ica-
ions o ood-g ade enzymes issued by he Join FAO/WHO Expe Commi ee on Food
Addi i es (JECFA) and he Food Chemicals Codex (FCC).
4.2. Expe imen al Design and S a is ical Analysis
Design o expe imen s (DOE) is a ool ha enables he examina ion o he in luence
o independen a iables (p ocess ac o s) on dependen a iables. The e o e, i enables
he iden i ica ion o signi ican ac o s o he p ocess and i s op imiza ion [
53
]. Va ious
expe imen planning designs a e used in p ac ice, e.g., h ee-le el ull ac o ial design,
cen al composi e design, Box-Behnken design, o Taguchi design [
54
]. The ex ac ion
empe a u e and ex ac ion ime p o ed o be key p ocess ac o s ha in luence no only he
deg ee o con e sion o collagen o gela ins bu also he p ope ies o gela ins. The e o e,
hese ac o s we e s udied using he Taguchi design o he expe imen s. This will achie e a
mo e e ec i e op imiza ion o he gela in p epa a ion p ocess om he MDCM by-p oduc .
Independen a iables wi h ac o le els: ac o A (ex ac ion empe a u e), 42, 46, 50
◦
C;
ac o B (ex ac ion ime), 20, 40, 60 min. The selec ed dependen a iables we e as ollows:
gela in yields (Y
G1
, Y
G2
, Y
G3
), gel s eng h, iscosi y, MP, GP, WHC, FBC, FC, FS, EC, and
ES.
The gela in analysis was pe o med in iplica e; mean alues we e calcula ed using
Mic oso O ice Excel 2013 (Mic oso , Den e , CO, USA). Mini ab
®
17.2.1 s a is ical so -
wa e o Windows (Fuji su L d., Tokyo, Japan) was used o pe o m eg ession analysis
o he da a ob ained. The s a is ical signi icance was e alua ed using analysis o a iance
(ANOVA). The le el o signi icance was es ablished a 5% (p- alue
≤
0.05); ac o s wi h a
alue <0.05 ha e an e ec on he p ocess a iables e alua ed wi h 95% p obabili y. The
same so wa e e alua ed he g aphical analysis o he da a by c ea ing con ou plo s show-
ing he ela ionship be ween he dependen a iables and he independen a iables by
iewing disc e e con ou s o he dependen esponse a iables.

In . J. Mol. Sci. 2023,24, 3654 17 o 22
4.3. P ocessing o MDCM By-P oduc in o Gela ins
The scheme o complex p ocessing o MDCM by-p oduc in o h ee ac ions o
gela ins, including usable by-p oduc s c ea ed du ing p ocessing, is shown in a low
cha in ou echnological sec ions, see Scheme 1.
In . J. Mol. Sci. 2023, 24, x FOR PEER REVIEW 18 o 23
Scheme 1. In ou case, we p oposed an inno a i e p ocess o dis up ing he qua e na y s uc u e o
pu i ied (demine alized) collagen: a p o eoly ic enzyme condi ioning and gela in ex ac ion in 3
s ages. The de ails o he p ocedu e a e gi en below.
I. Sepa a ion o o ganic ma e . The hawed aw ma e ial was i s washed wi h cold
H2O. I was mixed wi h 0.2 mol/L NaCl in a 1: 6 a io and shaken a oom empe a u e
(22.0 ± 1.0 °C) o 90 min and hen washed wi h cold H2O. I was hen mixed wi h 0.03
mol/L NaOH in a 1:6 a io and shaken a oom empe a u e o 45 min and, a e il a ion,
washed wi h cold H2O; his p ocedu e was epea ed h ee mo e imes. Finally, he aw
ma e ial was washed wi h cold H2O and d ied a 35 ° C o 24 h. This was ollowed by he
de a ing s ep: he aw ma e ial was mixed in a 1:9 (w/ ) a io wi h pe oleum e he and
e hanol (mixed in a a io o 1:1, / ) and shaken o 48 h a oom empe a u e; a e 12 h,
he sol en was changed.
II. Demine aliza ion. The aw ma e ial was mixed in a 1:8 a io wi h 3.0% HCl and
demine alized wi h gen le shaking a oom empe a u e o 96 h; a e 24 h, he acid was
eplaced wi h a new one. A e il a ion (mace a ion liquo as a by-p oduc o he
p ocess), he demine alized collagen was ho oughly washed wi h cold H2O and d ied o
24 h a 35 °C.
III. Pu i ied collagen was mixed wi h H2O in a 1:10 a io and a e shaking o 20 min,
he pH was adjus ed o 6.5–7.0 (by adding a 5% NaOH solu ion). The 0.6 % p o eoly ic
enzyme (based on he weigh o pu i ied collagen) was hen added and he mix u e was
shaken a oom empe a u e o 24 h; du ing he i s 4 h a 30-min in e als, he pH was
checked (and adjus ed) o he p esc ibed ange. A e il e ing o he liquid by-p oduc
(collagen hyd olysa e), solid collagen was washed ho oughly wi h cold H2O. Collagen
hyd olysa e was d ied in a hin laye (4 mm) in a ci cula ing ai d ie a 60.0 ± 0.5 °C o
20 h.
IV. 3-s ep ex ac ion o gela ins. Bio echnologically ea ed collagen was subjec ed o
3 sepa a e (sequen ial) ex ac ion cycles using a ba ch p ocess ex ac o . In he i s
ex ac ion s age, collagen was mixed wi h H2O in a a io o 1:20 and he mix u e was
hea ed while s i ing a a a e o d /dτ = 10 °C/min o a empe a u e acco ding o ac o A
(42.0 ± 0.5, 46.0 ± 0.5, 50.0 ± 0.5 °C), a which poin he gela in ex ac ion las ed o he ime
acco ding o ac o B (20, 40, 60 min). A e il a ion, he solu ion o he 1s gela in ac ion
was immedia ely hea ed o a empe a u e o 85.0 ± 0.5 °C (d /dτ = 15 °C/min) and kep a
his empe a u e o 8 min; he esidual enzyme was inac i a ed his way. The gela in
solu ion was pou ed in o a hin ilm (4 mm) and d ied in a ci cula ing ai d ie , i s a
Scheme 1.
In ou case, we p oposed an inno a i e p ocess o dis up ing he qua e na y s uc u e
o pu i ied (demine alized) collagen: a p o eoly ic enzyme condi ioning and gela in ex ac ion in
3 s ages. The de ails o he p ocedu e a e gi en below.
I. Sepa a ion o o ganic ma e . The hawed aw ma e ial was i s washed wi h cold
H
2
O. I was mixed wi h 0.2 mol/L NaCl in a 1: 6 a io and shaken a oom empe a-
u e (
22.0 ±1.0 ◦C
) o 90 min and hen washed wi h cold H
2
O. I was hen mixed wi h
0.03 mol/L NaOH in a 1:6 a io and shaken a oom empe a u e o 45 min and, a e
il a ion, washed wi h cold H
2
O; his p ocedu e was epea ed h ee mo e imes. Finally,
he aw ma e ial was washed wi h cold H
2
O and d ied a 35
◦
C o 24 h. This was ollowed
by he de a ing s ep: he aw ma e ial was mixed in a 1:9 (w/ ) a io wi h pe oleum e he
and e hanol (mixed in a a io o 1:1, / ) and shaken o 48 h a oom empe a u e; a e
12 h, he sol en was changed.
II. Demine aliza ion. The aw ma e ial was mixed in a 1:8 a io wi h 3.0% HCl and
demine alized wi h gen le shaking a oom empe a u e o 96 h; a e 24 h, he acid was
eplaced wi h a new one. A e il a ion (mace a ion liquo as a by-p oduc o he p ocess),
he demine alized collagen was ho oughly washed wi h cold H
2
O and d ied o 24 h a
35 ◦C.
III. Pu i ied collagen was mixed wi h H
2
O in a 1:10 a io and a e shaking o 20 min,
he pH was adjus ed o 6.5–7.0 (by adding a 5% NaOH solu ion). The 0.6 % p o eoly ic
enzyme (based on he weigh o pu i ied collagen) was hen added and he mix u e was
shaken a oom empe a u e o 24 h; du ing he i s 4 h a 30-min in e als, he pH was
checked (and adjus ed) o he p esc ibed ange. A e il e ing o he liquid by-p oduc
(collagen hyd olysa e), solid collagen was washed ho oughly wi h cold H
2
O. Collagen
hyd olysa e was d ied in a hin laye (4 mm) in a ci cula ing ai d ie a 60.0
±
0.5
◦
C o
20 h.
IV. 3-s ep ex ac ion o gela ins. Bio echnologically ea ed collagen was subjec ed
o 3 sepa a e (sequen ial) ex ac ion cycles using a ba ch p ocess ex ac o . In he i s
ex ac ion s age, collagen was mixed wi h H
2
O in a a io o 1:20 and he mix u e was
hea ed while s i ing a a a e o d /d
τ
= 10
◦
C/min o a empe a u e acco ding o ac o A
(
42.0 ±0.5
, 46.0
±
0.5, 50.0
±
0.5
◦
C), a which poin he gela in ex ac ion las ed o he
ime acco ding o ac o B (20, 40, 60 min). A e il a ion, he solu ion o he 1s gela in
In . J. Mol. Sci. 2023,24, 3654 18 o 22
ac ion was immedia ely hea ed o a empe a u e o 85.0
±
0.5
◦
C (d /d
τ
= 15
◦
C/min)
and kep a his empe a u e o 8 min; he esidual enzyme was inac i a ed his way. The
gela in solu ion was pou ed in o a hin ilm (4 mm) and d ied in a ci cula ing ai d ie , i s
a 40.0
±
0.5
◦
C o 12 h, and hen a 65.0
±
0.5
◦
C o 8 h. The esul ing gela in ilm was
sc aped, weighed, and g ound o a powde . In he second and hi d ex ac ion s ages, he
same p ocedu e was ollowed a ex ac ion empe a u es o 65.0
±
0.5
◦
C o 30 min and
80.0
±
0.5
◦
C o 30 min. The second gela in ac ion was inac i a ed in he same way as
he i s gela in ac ion. The undissol ed esidue (a by-p oduc o he ex ac ion o gela in)
emained a e he hi d ex ac ion cycle and was d ied a 103.0
±
1.0
◦
C o cons an weigh
and hen weighed. The p epa ed gela ins we e subjec ed o u he analysis.
4.4. Analy ical Pa
The hyd olysa e yield (Y
H
) was calcula ed om he weigh o he hyd olysa e p epa ed
a e condi ioning he pu i ied collagen acco ding o he ini ial weigh o he pu i ied
collagen (Equa ion (6)), he yield o gela ins (Y
G1
, Y
G2
, Y
G3
) om he weigh o ex ac ed
gela ins acco ding o he ini ial weigh o he pu i ied collagen (Equa ion (7)). Fu he mo e,
he o al ex ac ion yield o gela in (
Σ
Y
G
) and he po ion o undissol ed esidue (UR)
was calcula ed (Equa ions (8) and (9)). The mass balance e o (MBE) is exp essed by he
pe cen age di e ence o he d y ma e mass balance be ween he inpu (pu i ied collagen)
and he ou pu (hyd olysa e + gela ins + undissol ed esidue); see Equa ion (10).
YH= (mH/m0)×100 (6)
YG= (mG/m0)×100 (7)
ΣYG= YG1+YG2 + YG3 (8)
UR = (mUR/m0)×100 (9)
MBE = [100 −(YH+ YG1 + Y G2 + Y G3 + UR)] (10)
whe e Y
H
is he hyd olysa e yield (%), Y
G1
is he yield o he i s gela in ac ion (%), Y
G2
is he yield o he second gela in ac ion (%), Y
G3
is he yield o he hi d gela in ac ion
(%), UR is an undissol ed esidue (%), m
0
is he weigh o pu i ied collagen (g), m
H
is
he hyd olysa e weigh (g), m
G
is he weigh o gela ins (g), and m
UR
is he weigh o he
undissol ed esidue (g).
Gel s eng h, iscosi y, and ash con en we e de e mined acco ding o s anda d es
me hods o edible gela ins [
55
]. Because hese a e common gela in es ing me hods, we
p esen only hei p inciples. The gel s eng h was de e mined om a gel o med om
a 6.67 % solu ion p epa ed acco ding o p esc ibed condi ions by measu ing he o ce
(weigh in g ams, which is equal o he Bloom alue) equi ed o dep ess a p esc ibed a ea
o he sample su ace o a dis ance o 4 mm. The iscosi y o a 6.67 % gela in solu ion
was de e mined by he Ubbelohde iscome e and ash g a ime ically a e bu ning and
annealing he sample. The ollowing gela in p ope ies a e no desc ibed in s anda d gela in
es ing me hods, so a b ie es p ocedu e will be p o ided.
Gela in wa e holding capaci y was de e mined acco ding o Nas in e . al. [
56
] wi h
sligh modi ica ions. In a plas ic es ube, 1.0 g o he gela in sample was mixed wi h
25.0 mL o dis illed H
2
O and hen he con en s we e shaken igo ously o 5 min a oom
empe a u e. The con en s o he es ube we e hen cen i uged a 5000 pm o 30 min
and hen he supe na an was il e ed h ough Wha man no. 1. il e pape . WHC (%) was
calcula ed om he weigh o wa e abso bed by he gela in sample, w
1
(g), based on he
weigh o gela in weighed, w
0
(g), and mul iplied by a coe icien o 100; see Equa ion (11).
WHC = (w1/w0)×100 (11)
Gela in a binding capaci y was de e mined acco ding o Li e . al. [
57
]. In a plas ic
es ube, 0.1 g o he gela in sample was mixed wi h 10.0 mL o sun lowe oil, and he
In . J. Mol. Sci. 2023,24, 3654 19 o 22
con en s we e igo ously shaken o 30 min a oom empe a u e. The con en s o he es
ube we e hen cen i uged a 2500 pm o 30 min and he supe na an was pipe ed and
weighed. FBC (%) was calcula ed om he weigh o oil abso bed by he gela in sample,
w
2
(g), based on he weigh o gela in weighed, w
0
(g), and mul iplied by a coe icien o
1000; see Equa ion (12).
FBC = (w2/w0)×1000 (12)
Gela in oaming capaci y and oamings abili ywe e de e mined acco ding oSa hee .al.[
58
]
wi h sligh modi ica ions. The amoun o 1.0 g o he gela in sample was weighed in a g adua ed
cylinde and 50.0 mL o dis illed H
2
O was added; he gela in was dissol ed in a wa e ba h a
60.0
±
1.0
◦
C while s i ing. A e dissol ing, a dispe sing ins umen was placed below he le el
o he esul ing solu ion and he solu ion was whipped a 10,000 pm o 5 min. A e whipping,
he olume o he whipped solu ion was measu ed; FC (%) was calcula ed acco ding o Equa ion
(13). A e s anding a oom empe a u e o 30 min, he olume o he whipped solu ion was
measu ed again; FS (%) was calcula ed acco ding o Equa ion (14).
FC = [(V1−V0)/V0]×100 (13)
FS = [(V2−V0)/V0]×100 (14)
whe e V
0
is he o iginal olume o liquid (50 mL), V
1
is he olume o he whipped solu ion
(mL), and V2is he olume o he whipped solu ion a e 30 min (mL).
Gela in emulsi ying capaci y and he emulsion s abili y we e de e mined acco ding o
Ne o e . al. [
59
] wi h sligh modi ica ions. In a plas ic es ube, 0.01 g o he gela in sample
was mixed wi h 5.0 mL o dis illed H
2
O, and a e 10 s o ho ough shaking, 5.0 mL o
sun lowe oil was added and shaken o 1 min a oom empe a u e. The con en s o he
es ube we e hen cen i uged a 1000 pm o 5 min. The heigh s o he en i e olume
o liquid in he ube and he emulsion we e measu ed. The ube was hen placed in a
p ehea ed wa e ba h a 55.0
±
0.5
◦
C o 5 min; hen, he emulsion heigh was measu ed.
EC (%) and ES (%) we e calcula ed acco ding o Equa ions (15) and (16).
EC = (h1/h0)×100 (15)
ES = (h2/h0)×100 (16)
whe e h
0
is he heigh o he en i e olume o liquid (mm), h
1
is he heigh o he emulsion
a e cen i uga ion (mm), and h
2
is he heigh o he emulsion a e 5 min o hea ing (mm).
The Moosa i-Nasab me hod [
60
] wi h some modi ica ions was used o de e mine he
mel ing poin ; a solu ion o gela in a he same concen a ion (6.67%) as a e de e mina ion
o gel s eng h and iscosi y was used. A gela in solu ion was in oduced in o a glass
capilla y o 3.0 mm in diame e o o m a column a a heigh o 6.0
±
1.0 mm. The sample
capilla y was allowed o cool a 10.0
±
0.1
◦
C o 17 h (sol-gel ansi ion). The capilla y
was hen placed in a wa e ba h a 10.0
±
0.5
◦
C so i was comple ely imme sed. The wa e
ba h was hea ed a 2
◦
C/min and he gela in column in he capilla y was moni o ed. The
empe a u e a which he gela in column began o mo e in he capilla y (gel-sol ansi ion)
was eco ded as he MP.
The Sch iebe and Ga eis me hod [
2
] wi h sligh modi ica ions was used o de e mine
he gelling poin ; a gela in solu ion a he same concen a ion (6.67%) as a e de e mina ion
o gel s eng h and iscosi y was used. The gela in solu ion in he es ube was placed in a
wa e ba h. A e wa ming o 35.0
±
0.5
◦
C, ice wa e was added o he wa e ba h so ha
he cooling a e o he gela in solu ion in he ube was 2
◦
C/min. Each ime he empe a u e
d opped by 0.5
◦
C, a 0.10 g me al ball was inse ed in o he ube. The empe a u e a which
he ball go s uck in o on he gela in solu ion laye was eco ded as a GP.
In . J. Mol. Sci. 2023,24, 3654 20 o 22
5. Conclusions
The wo k is a con ibu ion o he esolu ion o issues o en i onmen al aspec s o
bioma e ials. I has been p o en ha wi h he app op ia e choice o inno a i e p ocessing
echnology using Taguchi design as a mode n me hod o expe imen planning, i is possible
o p epa e high-quali y gela ins om unused esidue a ising du ing he p oduc ion o
mechanically deboned chicken mea (MDCM). A comple ely new bene i o he wo k is
he e y high gela in yields, which ha e no been achie ed in p e ious wo ks dealing wi h
MDCM by-p oduc p ocessing. Fu he mo e, in e media e p oduc s o med du ing p o-
cessing do no ep esen esidual was e and can be u he used; he p esen ed echnology
belongs o ze o was e p ocessing o he MDCM by-p oduc . One o he p ac ical bene i s o
he wo k is ha , e en om a non adi ional sou ce o collagen, gela ins o di e en quali y
can be p epa ed by mul is age ex ac ion. These a e compa able o adi ional po k and
bee gela ins. Gela ins p epa ed om MDCM by-p oduc s a e sui able o common ood
and pha maceu ical applica ions, o cosme ic p oduc s, and o p oduc ion o biomedical
ma ixes as well.
Au ho Con ibu ions:
Concep ualiza ion, P.M.; me hodology, P.M.; so wa e, J.P.; alida ion, R.G.
and P.M.; o mal analysis, R.G.; in es iga ion, J.P.; esou ces, R.G.; da a cu a ion, P.M.; w i ing—
o iginal d a p epa a ion, P.M.; w i ing— e iew and edi ing, P.M.; isualiza ion, J.P.; supe ision,
P.M.; p ojec adminis a ion, R.G.; unding acquisi ion, R.G. All au ho s ha e ead and ag eed o he
published e sion o he manusc ip .
Funding: This esea ch ecei ed no ex e nal unding.
In o med Consen S a emen : No applicable.
Da a A ailabili y S a emen : The da a a e a ailable om he co esponding au ho .
Acknowledgmen s:
The au ho s hank Da id Dohnal (Pˇ e o , he Czech Republic) o edi ing he
manusc ip .
Con lic s o In e es : The au ho s decla e no con lic o in e es .
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