In e na ional Jou nal o Biological Mac omolecules 186 (2021) 365–376
A ailable online 8 July 2021
0141-8130/© 2021 The Au ho s. Published by Else ie B.V. This is an open access a icle unde he CC BY-NC-ND license
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E alua ion o easy- emo ing an ioxidan ilms o chi osan wi h Melaleuca
al e ni olia essen ial oil
Pa icia Caz´
on
a
, Aga a An oniewska
b
, Ja oslawa Ru kowska
b
, Manuel V´
azquez
c
,
*
a
Labo a o y o En i onmen al and Li e Sciences, No a Go ica Uni e si y, Slo enia
b
Ins i u e o Human Nu i ion Sciences, Facul y o Human Nu i ion, Wa saw Uni e si y o Li e Sciences, Nowou synowska s .159c, 02-776 Wa saw, Poland
c
Depa men o Analy ical Chemis y, Facul y o Ve e ina y, Uni e si y o San iago de Compos ela, 27002 Lugo, Spain
ARTICLE INFO
Keywo ds:
Film
Tea ee essen ial oil
Chi osan
ABSTRACT
Chi osan - ea ee essen ial oil (TTEO) ilms we e ob ained as a new biodeg adable ma e ial. Malic acid o lac ic
acid sol en s we e e alua ed o ob ain easy- emo ing ilms. The mic os uc u e by SEM and FT-IR, he he mal
p ope ies by TGA/DSC, he mechanical p ope ies, he wa e apo pe meabili y, he an ioxidan (DPPH
•
and
ABTS
•+
) ac i i y and he op ical p ope ies o he o mula ed ilms we e e alua ed. A comple e dissolu ion o he
ilm in wa e was ob ained. The elonga ion o b eak was highe in he ilms wi h malic acid (145.88–317.33%),
compa ing wi h hose wi h lac ic acid (25.54–44.08%). Chi osan ilm ob ained in malic acid wi h TTEO showed
he highes an ioxidan ac i i y. The colou and anspa ency o he samples did no su e signi ican a ia ions
by TTEO addi ion. Films showed good UV-ba ie p ope ies, wi h a sligh ly imp o emen by TTEO addi ion. The
ilms ob ained showed a g ea po en ial o ood packaging applica ions.
1. In oduc ion
Polysaccha ides such as chi osan can be a g ea al e na i e as poly-
me o educe he p esence o non-biodeg adable syn he ic packaging
[1–3].
Chi osan is a de i a e o chi in, one o he mos abundan biological
ma e ials in he wo ld. I is a high-molecula -weigh ca ionic na u al
polyme composed o andomly dis ibu ed chains o β-(1–4)D-glucos-
amine and N-ace yl-D-glucosamine [1]. Mos o he s udies on chi osan
highligh h ee main ac i e ea u es o chi osan ele an o packaging
pu pose: an imic obial, an ioxidan , and UV-ba ie p ope ies [4–7].
Mos s udies on chi osan ha e used ace ic acid as sol en o ob ain a
ilm- o ming solu ion [8–11]. Ne e heless, he p ope ies o he chi o-
san ma ix s ongly depend on he ype o sol en used. The e o e
changing he acid used o he elabo a ion o hese ilms will allow o
de elop no el ma ix wi h desi able p ope ies o ce ain applica ions
[12–14]. Fo example, he solubili y o he ilm can be modi ied using
o he ypes o sol en s such as malic o lac ic acid o ob ain a chi osan-
based ilm which a e easy o emo e om oods u . This is because
ace ic acid is ola ile and lac ic and malic acids a e no . They emain in
he ilm ma ix and imp o e solubili y in wa e . No e chi osan is soluble
in acid solu ions.
On he o he hand, na u al ex ac s om plan s, he bs o ui s a e a
p omising al e na i e o syn he ic p ese a i es. They can be included in
ac i e packaging. Besides, hese na u al ex ac s can sa is y consume
demand o na u al addi i es ollow he new end called “g een
consume ism” [8].
The essen ial oil o Melaleuca al e ni olia, also named as ea ee
essen ial oil (TTEO), is a complex mix u e wi h a majo sha e o
mono e penes, sesqui e penes, and hei alcohol de i a i es. The main
compounds o TTEO a e e pinen-4-ol, γ- e pinene,
α
- e pinene,
α
- e pineol,
α
- e pinolene and 1,8-cineole [15]. TTEO p esen s powe ul
an ioxidan p ope ies wi h wide applicabili y in he ood indus y.
Howe e , he di ec addi ion o hese kind o na u al an ioxidan s in o
oods u a e limi ed since he high hyd ophobic na u e and in ensi y o
he la ou associa ed o he ola ile compounds [16]. The an ibac e ial
p ope ies o ea ee oil a e well documen ed wi h iden i ica ion o
ac i e compounds agains bac e ia and yeas . Fo example, TTEO p e-
sen ed an imic obial ac i i y in g ound bee a he concen a ion 1.5%
/w agains Lis e ia monocy ogenes [17].
The use o polysaccha ide ma ices ha can be applied as ilm o
coa ing in ood a e an in e es ing al e na i e o se e as ehicles o
an ioxidan compounds such as TTEO [8].
P e ious s udy indica ed he an imic obial e ec i eness o chi osan-
* Co esponding au ho .
E-mail add ess: [email p o ec ed] (M. V´
azquez).
Con en s lis s a ailable a ScienceDi ec
In e na ional Jou nal o Biological Mac omolecules
jou nal homepage: www.else ie .com/loca e/ijbiomac
h ps://doi.o g/10.1016/j.ijbiomac.2021.07.035
Recei ed 2 June 2021; Recei ed in e ised o m 18 June 2021; Accep ed 4 July 2021
In e na ional Jou nal o Biological Mac omolecules 186 (2021) 365–376
366
based ilms ob ained in ace ic acid and en iched wi h TTEO agains
Lis e ia monocy ogenes, showing a comple e inhibi ion o he mic obial
g ow h du ing he i s i h days a 10 ◦C [8].
Despi e he po en ial applica ions o chi osan ilms wi h TTEO, no
s udies ha e been ound ha e alua e he in e ac ion o hese com-
pounds as an an ioxidan ac i e ma ix, o he in es iga ion o i s
unc ionali ies using sol en s o he han ace ic acid. A lack o a comple e
cha ac e iza ion o his ma e ial is e iden . The wa e solubili y p op-
e ies o apply as easy- emo ing an ioxidan ood packaging can be a
po en ial applica ion o TTEO-chi osan ilms.
The e o e, he p esen s udy was ocused on e alua ing he physi-
cochemical and unc ional p ope ies o essen ial oil o Melaleuca al e -
ni olia-chi osan ilms using malic o lac ic acid as sol en s. Soy leci hin
was e alua ed as na u al emulsi ie o achie e homogeneous dispe sion
o essen ial oil in aqueous solu ion. The wa e solubili y, equilib ium
mois u e con en , ensile p ope ies ( ensile s eng h, pe cen age o
elonga ion o b eak, Young's modulus and oughness), punc u e p op-
e ies (bu s s eng h and dis ance o bu s ), wa e apo pe meabili y
and an ioxidan p ope ies (DPPH
•
and ABTS
•+
) we e e alua ed. The
mic os uc u e, he mal and op ical p ope ies (UV ba ie , ans-
pa ency, opaci y and colou p ope ies) o he o mula ed biopolyme
we e assessed by scanning elec on mic oscopy, in a ed spec oscopy,
he mog a ime ic, di e en ial scanning calo ime y and UV–Vis
spec oscopy.
2. Ma e ials and me hods
Chi osan (M
w
100,000–300,000) we e pu chased om Ac os o -
ganics (Geel, Belgium), DL- malic acid ex a pu e and L(+)-lac ic acid
88–92% ex a pu e p o ided by Scha lau Mic obiology (Ba celona,
Spain), soy leci hin and pu e ea ee essen ial oil we e supplied by
Ko o S.L. (Alican e, Spain), we e used o p epa e he chi osan ilm-
o ming solu ion.
2.1. P epa a ion o ilms
Chi osan ilms (Ch- ilms) we e made by dissol ing 1% (w/w) chi o-
san in an aqueous solu ion o 2% (w/ ) malic acid o 2% ( / ) lac ic acid
(Table 1). The acid concen a ion was adjus ed o achie e he comple e
dissolu ion o chi osan, gi ing a c ys alline solu ion. Soy leci hin (0.1%
w/ ) was added in o chi osan solu ion as na u al emulsi ie agen . Chi-
osan solu ion - soy leci hin was homogenized a 15000 pm o 5 min
(Ul a Tu ax®, IKA, S au en, Ge many). The mix u e was degassed
using an ul asonic ba h o 15 min, ime enough o emo e he oam.
The TTEO was added un il a concen a ion anged be ween 0 and 1.0%
/ , and homogenized a 15,000 pm o 5 min ollowing by a degassed
s ep wi h ul asonic ba h o 15 min.
The esul ing ilm- o ming solu ion was cas ed in Pe i dish and d ied
o 48 h a oom empe a u e. The Ch- ilms we e cu o a speci ic size
and s o ed a de e mined condi ions depending on he es pe o ming.
The hickness (mm) was measu ed a i e andom loca ions using a
Thickness Me e ET115S (E a i GmbH, S u ga , Ge many).
2.2. Analysis o ola ile compounds o TTEO
Vola ile compounds o TTEO we e de e mined by headspace solid
phase mic o-ex ac ion (SPME) coupled wi h gas ch oma og aphy/mass
spec ome y (GC/MS) (6890N GC, 5975 MS Agilen , USA) wi h using
SPME ibe 50/30
μ
m (DVB/CAR/PDMS) (Supelco, Belle on e, PA,
USA). De ails o he ch oma og aphic sepa a ion and quan i a i e
analysis we e p e iously desc ibed [18].
2.3. Scanning elec on mic oscopy (SEM)
The mo phology o he Ch- ilms manu ac u ed in malic o lac ic acid
solu ions we e obse ed and analysed by SEM images. D ied and gold-
coa ed samples we e pho og aphed wi h a high- acuum mic oscope
(JEOL JSM-6360LV, Jeol L d., Tokyo, Japan) a an accele a ing ol age
o 20 kV. Samples we e a ached on slides using conduc i e double-sided
ca bon ape.
2.4. Fou ie ans o m in a ed spec oscopy (FT-IR)
FT-IR was ca ied ou using a FT-IR ABB Bomen 102 (ABB L d.,
Zu ich Swi ze land) equipped wi h a Uni e sal A enua ed To al
Re lec ance (UATR) accesso y (SPECAC Golden Ga e) wi h diamond
c ys al. P io o he es , he selec ed samples we e condi ioned a 65 ±
2% ela i e humidi y and 21 ±1 ◦C o 48 h. FTIR spec a we e eco ded
a a spec al esolu ion o 4 cm
−1
in he ange (400–4000) cm
−1
.
2.5. Wa e solubili y, equilib ium mois u e con en and wa e apo
pe meabili y
Wa e solubili y o he samples was con i med using he g a ime ic
me hod o imme sion in a de e mined olume o dis illed wa e as e-
po ed elsewhe e [19]. The equilib ium mois u e con en (%W) was
calcula ed ollowing he g a ime ic me hod by measu ing he di e -
ences be ween he weigh s o condi ioned (a 57% ela i e humidi y, %
RH) and d ied samples as epo ed elsewhe e by Cazon e al. [20]. Wa e
apo pe meabili y (WVP) we e measu ed ollowing he ASTM S anda d
Tes Me hod E96 as desc ibed elsewhe e [20]. The WVP o he samples
we e measu ed a 30 ◦C and conside ing an in e media e wa e ac i i y
alue be ween he wo le els which de ine he ela i e humidi y (RH)
g adien used in WVP de e mina ion [21]. The es was un a leas 8 h,
enough ime o each a dynamic equilib ium in he wa e lux. Each es
was pe o med by iplica e.
2.6. Mechanical p ope ies analysis
The mechanical p ope ies o he de eloped ilms we e analysed by
ensile and punc u e es s using a ex u ome e (TA-XTplus, S able Mic o
Sys em, UK) wi h he accesso ies indica ed o each es . Tensile s eng h
(TS, MPa), pe cen age o elonga ion a b eak (%E, %), Young's Modulus
(YM, MPa), oughness (T, MJ/m
3
), bu s s eng h (BS, g) and dis ance o
bu s (DB, mm) we e measu ed as desc ibed elsewhe e [20,22]. The
ensile es was ca ied ou ollowing he s anda d me hod D-882
(ASTM) and se ing he jaw sepa a ion in 20 mm and he a e o loading
in 1 mm/s.
2.7. An ioxidan p ope ies
The an ioxida i e capaci y o he de eloped ilms and i s imp o e-
men by he addi ion o TTEO was de e mined by wo me hods: 1,1-
diphenyl-2-pic ylhyd azyl adicals (DPPH
•
) and 2,2′-azino-bis (3-e h-
ylbenzo hiazoline-6-sul onic acid) (ABTS
•+
) ee adical sca enging
Table 1
Chi osan ilms o mula ion.
Film samples Chi osan O ganic acid Leci hin TTEO
% (w/w) M: % (w/ )
L: % ( / )
% (w/ ) % ( / )
Chi osan in malic acid solu ion
CH_M1 1 2 0 0
CH_M2 1 2 0.1 0
CH_M3 1 2 0.1 0.5
CH_M4 1 2 0.1 1.0
Chi osan in lac ic acid solu ion
CH_L1 1 2 0 0
CH_L2 1 2 0.1 0
CH_L3 1 2 0.1 0.5
CH_L4 1 2 0.1 1.0
CH - chi osan; M - malic acid; L - lac ic acid; TTEO - ea ee essen ial oil.
P. Caz´
on e al.
In e na ional Jou nal o Biological Mac omolecules 186 (2021) 365–376
367
assay. The aliquo s we e ob ained by he me hanolic ex ac s o he
ilms, using 0.4 g o sample in 24 mL o e hanol and le o e nigh in
da kness.
Radical sca enging ac i i y o he Ch- ilms on DPPH
•
was analysed
using a UV–Vis spec opho ome e (Speco d 40, Analy ik Jena AG,
Ge many) a 515 nm ollowing he me hod desc ibed elsewhe e [23].
The ABTS
•+
adical ca ion sca enging ac i i y o he Ch- ilm samples
was measu ed spec opho ome ically measu ing he abso bance a 734
nm ollowing he me hod desc ibed elsewhe e [24]. The esul s we e
exp essed as %DPPH
•
and %ABTS
•+
sca enging ac i i y.
2.8. Op ical and he mal p ope ies o ilms (TGA/DSC)
The UV–Vis spec a o he samples we e pe o med by spec opho-
ome e V-670 (Jasco Inc., Japan) in he UV–Vis ligh egions (190
nm–800 nm), se led 2 nm in e als. The ilms we e cu in ec angula
pieces and placed inside he es cell o he spec opho ome e . The es
was un in duplica e o each sample. The anspa ency and opaci y
alues o he samples we e calcula ed om he ansmi ance and
abso bance alues o he samples a 500 nm and 600 nm o wa eleng h,
as desc ibed elsewhe e [19]. The CIE L*a*b* coo dina es we e de e -
mined by he Spec a Manage so wa e (Jasco Inc., Japan) es ablished
on he second s anda d obse e wi h ligh sou ce D65.
A he mog a ime y and di e en ial scanning calo ime y equip-
men TGA/DSC (Me le Toledo, Swi ze land) we e used o analyse he
he mal p ope ies and s abili y. The samples we e placed in he me ic
aluminium pans and he es was ca ied ou a a hea ing a e o 10 ◦C/
min om 50 o 400 ◦C, in a mosphe e o N
2
(50 mL/min).
2.9. S a is ical analysis
Resul ob ained we e s a is ically analysed by one-way analysis o
a iance (ANOVA) employing Mic oso Excel® so wa e. Di e ences
be ween pai s o means we e assessed based on con idence in e als
using he Tukey Pos Hoc es . The leas signi icance di e ence was p <
0.05.
3. Resul s and discussion
3.1. Vola ile composi ion o TTEO
The analysis o ola ile compounds in he headspace o TTEO using
SPME yielded a o al o 36 iden i ied and quan i ied compounds
(Table S1). Mono e penes we e he mos abundan (87.3%) wi h 14
compounds iden i ied. Wi h acco dance o li e a u e, e pinen-4-ol was
a p edominan compound and accoun ed o 25.5% o he o al peak
a ea [25]. The o he quan i a i ely impo an compounds also included
in he mono e pene g oup: γ- e pinene (18.5%),
α
- e pinene (17.1%),
α
-pinene (8.04%),
α
- e pinolene (5.7%), m-cymene (4.7%) and sabinene
(4.5%). All iden i ied mono e penes a e known as TTEO ma ke com-
pounds and con ibu ed o an ioxidan and an imic obial ac i i y o
TTEO [15]; [25]. Six een sesqui e penes we e iden i ied in mino sha e
(9.4%).
3.2. Film cha ac e iza ion
The ilm- o ming solu ion was p epa ed a oom empe a u e
ob aining a low ene gy demand p ocess. An acid concen a ion up o 2%
was needed o each a comple e dissolu ion o chi osan. In he lac ic acid
solu ions, a signi ican amoun o oam was o med du ing he s i ing
p ocess o chi osan and du ing he homogeneous dispe sion o soy
leci hin and essen ial oil. The oam was easily emo ed unde ul asound
ea men , gi ing a anspa en solu ion ee o ai bubbles. The addi ion
o soy leci hin and essen ial oil esul ed in solu ions wi h a milky
appea ance, as shown Fig. 1a. Despi e he whi ish appea ance o he
chi osan-leci hin-essen ial oil solu ion (Fig. 1b), once he wa e has
Fig. 1. Visual appea ance o he chi osan ilm- o ming solu ion wi h and
wi hou soy leci hin and ea ee essen ial oil (A) o he we ilms (B) and d ied
ilms (C) wi h malic acid.
P. Caz´
on e al.
In e na ional Jou nal o Biological Mac omolecules 186 (2021) 365–376
368
e apo a ed du ing he cas ing p ocess, he inal ilm ob ained did no
ha e he whi ish colou o he solu ion and shows he anspa en colou
o pu e chi osan samples, as shown Fig. 1c.
The ilms o mula ed om lac ic acid we e peeled o and handle
easily a e d ying. The ilms ob ained om malic acid we e mo e ad-
hesi e. This adhesi e cha ac e is ic o chi osan-based ilms om malic
acid could be a g ea ad an age o be applied easily as ilm o coa ing on
ood.
The o mula ions o he expe imen s a e shown in Table 1. The
a e age hickness o he samples anged be ween 4.31⋅10
−2
and
5.18⋅10
−2
mm.
3.3. Scanning elec on mic oscopy (SEM)
The SEM images o he su ace and c oss sec ion (Fig. 2) o he
de eloped Ch- ilms showed a homogeneous dispe sion o he soy leci-
hin and TTEO. The SEM images showed a smoo h su ace wi hou he
p esence o c acks o dis up ions due o he p esence o TTEO
h oughou he ma ix. In addi ion, he absence o c acks in he ilms
om lac ic acid solu ions (Fig. 2b–d) con i ms ha he ul asound
ea men was su icien o emo e he excess bubbles p oduced unde
agi a ion.
The c oss-sec ion SEM images (Fig. 2c–d) showed a compac s uc-
u e o he Ch- ilms p oduced om bo h acid sol en s. No e he di e -
ence in cu -o be ween ilms om he malic acid solu ions (Fig. 3c) and
lac ic acid (Fig. 3d). SEM images o ilms wi h lac ic acid showed a clean
cu , unlike he c oss sec ion o ilms wi h malic acid. The c oss sec ion o
samples wi h malic acid showed a ubbe y beha iou . This beha iou o
he ilms wi h malic acid concu ed wi h he adhesi e beha iou
obse ed du ing he handling o he samples.
In li e a u e, SEM images o chi osan-based ilms elabo a ed om
ace ic acid solu ions and en iched wi h TTEO wi hou any emulsi ie
showed a discon inuous s uc u e o med by lipid d ople s embedded in
he con inuous chi osan ne wo k [8]. The e o e, he addi ion o small
amoun s o soy leci hin (0.1% w/ ) achie ed a homogeneous dispe sion
o he essen ial oil in he ma ix. Soy leci hin p e en ed he agglome -
a ion o oil molecules in o la ge d ople s ha could cause a g ea e
dis up ion in he chi osan ne wo k.
3.4. Fou ie ans o m in a ed spec oscopy (FT-IR)
The FT-IR spec a o pu e chi osan samples ob ained om malic acid
and lac ic acid solu ions in he ange be ween 4000 and 800 cm
−1
a e
shown in Fig. 4a. FT-IR spec a o chi osan-based ilms wi h soy leci hin
and TTEO using malic acid and lac ic acid a e also shown in Fig. 3.
All spec a showed he cha ac e is ic abso bance peaks o he pu e
Ch- ilms. The b oad abso p ion band be ween 3500 and 3200 cm
−1
co esponded o he o e lapping o he s e ching ib a ion o hyd oxyl
g oups (
–
OH) and he asymme ic and symme ic s e ching o he
N
–
H bonds in he amino g oup o he chi osan [11]; [14]. The ab-
so p ion peaks a 2928 cm
−1
co esponded o he s e ching ib a ions
o he C
–
H bond in -CH
2
g oup [26]. The abso p ion peaks a 1647
cm
−1
and 1554 cm
−1
we e a ibu ed o C
–
–
O s e ching (amide I) and
N
–
H bending (amide II), espec i ely [27].
The main di e ences be ween he spec a o samples om malic o
lac ic acid we e ound in he egion om 2000 o 1000 cm
−1
. Simila
esul was obse ed in samples elabo a ed om lac ic acid and ace ic
acid [28]. The highe peaks shi ed in ilms om malic acid in he
2000–1000 cm
−1
ange we e due o he a ia ions in he p o ona ed
amine concen a ion o he samples. The in e ac ion be ween he sol en
acid and chi osan inc eases as he pK
a
o he acid dec eases. The pK
a
o
malic and lac ic acid a e 3.40 and 3.86, espec i ely, obse ing lowe
abso bance peaks o samples wi h lac ic acid [28].
The spec a o he samples en iched wi h TTEO showed he cha ac-
e is ic peaks o he pu e Ch- ilms wi h sligh shi s and lowe adso p-
ion. These changes we e p obably due o he o ma ion o new
Fig. 2. Scanning elec on mic oscopy images o chi osan ilms wi h ea ee essen ial oil 1.0% ( / ). Expe imen s a e de ined in Table 1.
P. Caz´
on e al.
In e na ional Jou nal o Biological Mac omolecules 186 (2021) 365–376
369
hyd ogen bonds be ween he -OH g oups om compounds in TTEO
(mos ly om e pinen-4-ol) and he –NH
3+
and -OH g oups in chi osan.
This obse a ion leads o he assump ion ha he e could be an
a angemen and in e ac ion in he ilm. Ch- ilms en iched wi h
u me ic ex ac [26], ci onella essen ial oil and ceda wood oil [27] and
osema y essen ial oil [11] showed a simila pa e n.
3.5. E alua ion o he wa e solubili y and equilib ium mois u e con en
The soluble ma e o he samples was analysed o de e mine he ease
o emo al o TTEO-chi osan ilms o coa ings. When he samples we e
subme ged in he con aine s wi h dis illed wa e , an ins an aneous
swelling o he sample was obse ed ollowed by he comple e loss o he
s uc u e. The esul s indica ed 100% dissolu ion o he Ch- ilms made
wi h malic acid o lac ic acid. Addi ion o TTEO did no dec ease he
solubili y o he samples.
Ch- ilms wi h lac ic acid sol en dissol ed comple ely in wa e
wi hin 24 h. The high solubili y o Ch- ilms wi h malic acid was no
p e iously epo ed. The comple e dissolu ion o chi osan-glyce ol
samples using lac ic acid as sol en was also obse ed [14]. These e-
sul s compa ed e y well wi h ha o chi osan ilms using ace ic acid as
sol en we e a wa e solubili y o only 11.39% was ob ained [29].
Mois u e con en (%W) o he equilib a ed ilms a s anda d condi-
ions was analysed. P e ious s udies demons a ed he ele an plas i-
cizing e ec o he wa e molecules on he inne s uc u e o
polysaccha ide-based polyme s, and consequen ly on hei unc ional
Fig. 3. FT-IR spec a o chi osan-based ilm wi h ea ee essen ial oil. A) FT-IR spec a o chi osan (CHI) samples using malic acid (CHI_M1) and lac ic acid (CHI_L1)
as sol en . B) FT-IR spec a o pu e chi osan (CHI_M1) and wi h 1% ea ee essen ial oil (CHI_M4) samples using malic acid as sol en . C) FT-IR spec a o pu e
chi osan (CHI_L1) and wi h 1% ea ee essen ial oil (CHI_L4) samples using lac ic acid as sol en .
P. Caz´
on e al.
In e na ional Jou nal o Biological Mac omolecules 186 (2021) 365–376
370
p ope ies [30,31].
As shown in Table 2, inclusion o TTEO in o CH- ilms did no showed
a signi ican in luence on he %W alues (p <0.05). Resul s indica ed
ha supplemen a ion CH- ilms wi h hyd ophobic TTEO (up o 1%) does
no educe he wa e -chi osan in e ac ions which would esul in a
disa angemen o ilm ne wo k. Unlike, adding osema y essen ial oil
up o 1.5% (w/w) o chi osan ilms p oduced a signi ican inc ease o
mois u e con en o he samples [11].
The mois u e con en o he samples s ongly depended on he ype
o acid used in he ilm- o ming solu ions (p <0.05), which in luenced
o he p ope ies. The di e ence in he mois u e was due o he join
e ec o se e al ac o s. Fi s , he chemical s uc u e di e ences o he
acids used [15]. The p o ona ion deg ee o he amide g oup o he chi-
osan and he p esence o mo e hyd oxyl g oups in lac ic acid p o ided
highe amoun o a ailable ac i e si es o wa e molecule in e ac ions
[14]. Fu he mo e, he in e molecula a angemen o chi osan in an
aqueous solu ion is in luenced by ionic s eng h and deg ee o dissoci-
a ion. The ype o acid may a ec bo h he densi y o he bonds and he
opological limi a ions o he ilm. Di e en in e ac ions be ween
chi osan-acid a e ep esen ed by he spa ial con igu a ion o he chi o-
san molecules du ing ilm o ma ion. These s uc u al changes will
a ec he inal p ope ies o he samples [13]. Rhim e al. [12] also
obse ed highe mois u e con en on chi osan-based ilm om lac ic
acid solu ion han om malic acid solu ions.
3.6. E alua ion o he wa e apo pe meabili y (WVP)
The WVP alues calcula ed o Ch- ilms using malic acid as sol en
we e anged be ween 3.88 ⋅10
−11
- 4.38 ⋅10
−11
g/m⋅s⋅Pa o samples
using malic acid and be ween 4.86 ⋅10
−11
- 6.16 ⋅10
−11
g/m⋅s⋅Pa o
samples using lac ic acid as sol en , as shown Table 2. The addi ion o
TTEO did no showed a signi ican in luence on WVP alues o ilms (p <
0.05). Lowe alues o WVP was obse ed in Ch- ilms combined wi h
bac e ial cellulose and glyce ol [32]. The non-signi ican in luence o
he TTEO concen a ion on WVP beha iou could be ela ed wi h he
low le els used. The hyd ophobic na u e o he essen ial oil was no
su icien o educe he pe meabili y o he ilm, which was p obably
also a ou ed by he in e nal s uc u al changes in he ma ix p omo ed
by he p esence o TTEO molecules.
P e ious wo ks indica ed ha highe oil phase a io o educed oil
pa icle size hinde ed he di usion o wa e molecules h ough he
ma ix [33]. TTEO up o 2% (w/w) showed a signi ican dec ease o he
WVP alues o chi osan ilm elabo a ed om ace ic acid solu ions [8].
Simila esul s o WVP alues as in ou s udy we e assayed in he
en iched wi h essen ial oils Ch- ilms o mula ed wi h ace ic acid [33]. I
should be also no ed ha Ch- ilms wi h lac ic acid showed sligh ly
highe WVP alues han wi h malic acid (Table 2).
As a o emen ioned in he FT-IR spec a sec ion, i can be ela ed wi h
he p ope ies o malic acid wi h mo e in e ac ions be ween chi osan-
malic acid han chi osan-lac ic acid. I was obse ed ha a lowe de-
g ee o in e ac ion be ween he componen s o he ma ix (glyce ol and
chi osan) causes easie mig a ion o wa e apo molecules h ough he
Fig. 4. UV-VIS spec a p o ile o A) Chi osan ilms wi h ea ee essen ial oil, using malic acid as sol en . B) Chi osan ilms wi h ea ee essen ial oil, using lac ic acid
as sol en . CHI, chi osan; L, lac ic acid and M, malic acid.
P. Caz´
on e al.
In e na ional Jou nal o Biological Mac omolecules 186 (2021) 365–376
371
ilm [29].
A sligh ly lowe WVP alues o Ch- ilms p epa ed wi h malic acid
han wi h lac ic acid was also epo ed [12,13]. The esul s ob ained in
hese s udies we e 2 o de o magni ude lowe han hose o he p esen
s udy. The WVP alues o Ch- ilms a e a ec ed by se e al ac o s, such
as chi osan p ope ies (molecula weigh , deace yla ion deg ee), ilm
composi ion (chi osan, sol en ype and/o plas icizing concen a ion),
WVP measu ing me hod and condi ions ( empe a u e, RH), co ec ion o
ai gap e ec and ilm hickness [12]. Thus, he WVP alues in he
li e a u e should be compa ed wi h cau ion, aking in o accoun all hese
ac o s.
3.7. E alua ion o he mechanical p ope ies
The esul s o mechanical p ope ies o Ch- ilms calcula ed by he
ensile and punc u e es s a e p esen ed in Table 2. The ensile es o
Ch- ilms wi h malic acid (CH_M1 and CH_M2) showed alues in he
anges 1.54–4.23 MPa o TS, 145.88–317.33% o %E, 1.81–2.96 MPa
o YM and 2.27–4.07 MJ/m
3
o T. Inclusion o he TTEO in he ilm
o mula ion had a signi ican e ec (p <0.05) on he ensile pa ame e s.
Subsequen ly, he Tukey pos hoc es de ec ed signi ican di e ences o
he means (p <0.05) a he highes TTEO concen a ion.
Using lac ic acid as sol en , Ch- ilms (CH_L1 and CH_L2) anged
4.09–6.32 MPa o TS, 25.54–44.08% o %E, 23.01–33.96 MPa o YM
and 0.81–1.74 MJ/m
3
o T. Simila ly like in he ilms wi h malic acid,
addi ion o TTEO signi ican ly in luenced he ensile pa ame e s o he
ilms wi h lac ic acid (Table 2).
The esul s showed he dec ease in esis ance o b eak and he in-
c ease in de o ma ion, dec easing he TS and YM alues and inc easing
he %E and T alues in ilms con ained 1% TTEO. This beha iou
esponded o a plas icizing e ec , indica ing ha addi ion o TTEO up o
1% ( / ) p omo ed a plas icizing e ec on he chi osan ne wo k.
The plas icizing e ec could be explained by he pa ial eplacemen
o s onge polyme -polyme in e ac ions by weake polyme -essen ial
oil in e ac ions in he ilm ne wo k. The new o med in e ac ions may
educe he cohesion o he polyme ne wo k o ces, dec easing he TS
and YM alues and inc easing he %E and T alues. The plas icizing
e ec is s ongly de e mined by he essen ial oil a io in he ma ix and
he sol en used o ob ain he chi osan ilm- o ming solu ion. The
bene icial plas icizing esul was achie ed wi h using malic acid solu ion
and highe TTEO concen a ions.
The pa ame e ha a ac ed he mos a en ion was %E, gi ing
alues be ween 4 and 10 imes highe o ilms made wi h malic han
wi h lac ic acid. This phenomenon is due o how chi osan chains in e ac
and he acid sol en ole. Chi osan ne wo k is o med by he hyd ogen
bonds be ween hyd oxyl g oups and amino g oups in he polyme
chains. These in e ac ions inc ease wi h he inc easing amoun o amino
and hyd oxyl g oups. Fo ha eason, he in e molecula a angemen o
chi osan in an aqueous solu ion is in luenced by he p ope ies o he
acid solu ions, such as ionic s eng h, deg ee o dissocia ion and coun e
ion s uc u e. Besides, di e en in e ac ions a e ep esen ed by he
spa ial con igu a ion o he chi osan molecules du ing ilm o ma ion
[12–14]. Consequen ly, he e ec o he oil and how i in e ac ed wi h
he polyme chains, depended on he sol en used, which de e mined
he in e ac ions be ween polyme chains and he con o ma ion o he
chi osan ne wo k.
The phospholipid composi ions o he soy leci hin could con ibu e
o he nega i e cha ge o he chi osan solu ion. The inclusion o phos-
pholipids in o chi osan solu ion could con ibu e o o ma ion he
su ace-ac i e amphiphilic molecules. The anionic cha ge may in e ac
wi h he posi i e cha ge o p o ona ed chi osan in acid solu ion and
modi y he chi osan ne wo k [16]. The plas icizing e ec o oils also
depends on he p esence o absence o emulsi ying agen s which allow
homogeneous dispe sion and a oid he o ma ion o la ge d ople s. Ch-
ilms en iched wi h ci onella essen ial oil, ceda wood oil [27], Za a ia
mul i lo a Boiss essen ial oil [34] showed a plas icizing e ec up o 10%
o oil concen a ion. [27] epo ed ha a highe essen ial oil concen-
a ions, he mechanical alues d opped by an excessi e subs i u ion o
polyme -polyme bonds by polyme -essen ial oil bonds, gi ing weak
s uc u es wi h lowe de o ma ion capaci y.
Ch- ilms en iched wi h TTEO o be gamo essen ial oil ilms wi hou
emulsi ying agen showed a signi ican dec ease in ensile s eng h,
de o ma ion a b eak and elas ic modulus. The au ho s jus i y he gen-
e al dec ease in all mechanical p ope ies due o he no homogeneous
Table 2
Physical and mechanical p ope ies o chi osan ilms.
Film
samples
%W TS %E YM
% MPa % MPa
CH_M1 20.34 ±0.31 4.23 ±0.69
a
158.80 ±8.93
a
2.85 ±0.21
a
CH_M2 19.98 ±0.57 3.40 ±0.24
a
145.88 ±
27.40
a
2.95 ±0.39
a
CH_M3 19.41 ±0.55 3.51 ±0.72
a
150.55 ±
25.10
a
2.96 ±0.28
a
CH_M4 19.26 ±0.82 1.54 ±
0.30
b
317.33 ±
22.84
b
1.81 ±0.22
b
CH_L1 59.88 ±0.65 6.32 ±2.29
c
38.30 ±12.04
c
33.96 ±5.06
c
CH_L2 60.14 ±3.15 6.15 ±0.91
c
44.08 ±7.37
c
33.72 ±6.94
c
CH_L3 59.37 ±0.90 4.40 ±
1.11
d
25.54 ±7.30
d
29.40 ±
3.04
cd
CH_L4 58.68 ±2.28 4.09 ±
0.61
d
33.10 ±3.08
cd
23.01 ±1.95
d
Film
samples
T BS DB WVP
MJ/m
3
g mm g/m⋅s⋅Pa
CH_M1 3.12 ±
0.74
ab
351.84 ±
23.20
a
5.41 ±
0.31
3.88⋅10
−11
±
9.58⋅10
−13
CH_M2 2.30 ±
0.64
a
347.27 ±
46.57
a
5.72 ±
1.14
4.38⋅10
−11
±
3.67⋅10
−12
CH_M3 2.27 ±
0.99
a
175.64 ±
20.06
b
5.09 ±
0.11
4.42⋅10
−11
±
4.73⋅10
−12
CH_M4 4.07 ±
1.43
b
199.90 ±
39.51
b
5.62 ±
0.90
4.30⋅10
−11
±
2.57⋅10
−12
CH_L1 1.74 ±
0.90
c
434.58 ±
28.52
c
4.04 ±
0.20
c
5.11⋅10
−11
±
6.04⋅10
−12
CH_L2 1.94 ±
0.49
c
360.52 ±
0.05
d
3.56 ±
0.20
c
6.16⋅10
−11
±
5.47⋅10
−12
CH_L3 0.81 ±
0.31
d
438.80 ±
13.59
c
3.65 ±
0.18
d
4.89⋅10
−11
±
1.19⋅10
−11
CH_L4 0.96 ±
0.11
d
358.05 ±
28.78
d
3.42 ±
0.22
d
4.86⋅10
−11
±
2.82⋅10
−13
CH - chi osan; M - malic acid; L - lac ic acid; %W - equilib ium mois u e con en ;
TS - ensile s eng h; %E - pe cen age o elonga ion o b eak; YM - Young's
modulus; T - oughness; BS - bu s s eng h (punc u e p ope ies); DB - dis ance
o bu s (punc u e p ope ies); WVP - wa e apo pe meabili y p ope ies.
Values a e exp essed as mean ±s anda d de ia ion (SD). Di e en le e s in he
same column indica e signi ican di e ences (p ˂ 0.05). Expe imen s a e de ined
in Table 1.
Table 3
An ioxidan p ope ies o he de eloped chi osan-based ilms.
Film samples Radical sca enging ac i i y on DPPH
•
and ABTS
•+
DPPH
•
(%) ABTS
•+
(%)
CH_M1 5.06 ±1.59
a
1.31 ±0.99
a
CH_M2 5.23 ±0.79
a
1.93 ±0.75
a
CH_M3 30.71 ±1.35
b
11.02 ±0.42
b
CH_M4 61.24 ±0.64
c
25.41 ±0.49
c
CH_L1 0.57 ±0.41
d
3.30 ±1.38
d
CH_L2 0.75 ±0.40
d
2.18 ±0.51
d
CH_L3 4.73 ±1.62
e
3.39 ±0.94
d
CH_L4 8.31 ±2.12
7.57 ±1.63
e
CH - chi osan; M - malic acid; L - lac ic acid; TTEO - ea ee essen ial oil.
Values a e exp essed as mean ±s anda d de ia ion (SD).
Di e en le e s in he same column indica e signi ican di e ences (p ˂ 0.05).
Expe imen s a e de ined in Table 1.
P. Caz´
on e al.
In e na ional Jou nal o Biological Mac omolecules 186 (2021) 365–376
372
a angemen o he lipid phase in he polyme ic ma ix [8,35].
The alues ob ained by he punc u e es showed a signi ican e ec
o TTEO addi ion on alues o BS in Ch- ilms wi h malic acid (Table 2).
Addi ion o TTEO dec eased he alues BS. The e we e no such clea
esul s o BS in Ch- ilms wi h lac ic acid. The de o ma ion su e ed by he
samples and how he polyme chains mus es uc u e unde he p es-
su e o o ces wi h di e en di ec ions is di e en om he es uc u ing
and de o ma ion unde ho izon al o ces. Fo his eason, he same in-
e ac ions wi hin he ma ix can show di e en esis ance and de o -
ma ion beha iou unde ensile o punc u e o ces [36].
3.8. An ioxidan p ope ies
Table 3 shows he an ioxidan ac i i y o all Ch- ilms de e mined by
wo me hods: ABTS
•+
and DPPH
•
, which allow o assay he capaci y o
sca enge ee adicals. As i was epo ed in p e ious wo ks, all pu e Ch-
ilms (CH_M1, CH_M2, CH_L1 and CH_L2) showed an ioxidan ac i i y in
bo h me hods, ega dless o he p esence o an emulsi ie [9,34].
The an ioxidan ac i i y o he CH_M1 and CH_L1 samples ep e-
sen ed 1.31 and 3.3% inhibi ion by ABTS
•+
me hod, espec i ely. This
ac i i y can be a ibu ed o he capaci y o chi osan amino g oups o
eac wi h ee adicals o o m ammonium g oups (NH
3
+
). The sca -
enging ac i i y o hese samples migh also be p o ided by he an ioxi-
dan p ope ies o he malic and lac ic acid [37,38]. The sol en used o
ob ain he ilm- o ming solu ion has a di ec e ec on he chi osan
p o ona ion and he ma ix con o ma ion, a ec ing he an ioxidan
p ope ies o pu e and en iched ilms.
A e adding TTEO, he an ioxidan ac i i y o ilms was subs an ially
inc eased. The deg ee o an ioxidan capaci y o supplemen ed ilms was
p opo ional o he amoun o TTEO added (0.5 and 1%). The well-
known an ioxidan p ope ies o TTEO a e a ibu ed o e penic com-
pounds which subs an ial amoun con i med in ou s udy (Table S1).
They can emain in he chi osan ma ix and p o ided he imp o ed
an ioxidan p ope ies o he ilm. The ob ained esul s a e in acco -
dance wi h he inc easing sca enging ac i i ies obse ed in Ch- ilms
wi h Eucalyp us globulus essen ial oil [9].
I espec i ely o he kind o o ganic acid in o mula ion, he DPPH
•
sca enging a es o TTEO supplemen ed ilms we e abou 6.5–12- old
highe han ha o pu e ilms (Table 3). Howe e , such spec acula
e ec in inc easing o an ioxidan ac i i y a e adding TTEO, assayed by
sca enging o ABTS
•+
ee adicals, was s a ed only in he case o ilms
wi h malic acid. I is necessa y o no e, ha en iched wi h TTEO Ch- ilms
con ained malic acid we e dis inguished by 6.5–7.5 highe DPPH
•
sca enging capaci y han ha con ained lac ic acid. I can be explained
by p o ed syne gic e ec o malic acid as p omo e agen s o an ioxi-
dan ac i i y o e pens [39].
3.9. E alua ion o he op ical p ope ies
Op ical p ope ies o he Ch- ilms we e e alua ed h ough he
ansmi ance p ope ies in he UV egion. The colou , anspa ency and
opaci y alues we e calcula ed om he ansmi ance alues in he Vis
egion. These pa ame e s p o ide use ul in o ma ion ega ding he po-
en ial applica ions o hese ilms o p o ec ood agains UV-ligh ,
keeping anspa ency high and opaci y low. Fo mula ion o poly-
saccha ide ilms wi h UV blocking p ope ies may be an e ec i e ool o
p e en nega i e e ec on la ou quali y, ligh -sensi i e nu ien s and
loss o i amins. The di ec UV ligh on ood migh lead o he p omp ing
o a se ies o eac ions ha a e ini ia ed by he elease o single oxygen,
causing discolo a ion, nu ien losses, o - la ou s and pho ooxida ion o
lipids and i amins [40,41].
The a e age pe cen age o ansmi ance (%T) in he UV-C (200–280
nm), UV-B (280–315 nm) and UV-A (315–400 nm) egions o he s udied
Ch- ilms a e shown in Table 4. The UV p o ile spec a o he ilms a e
shown in Fig. 4.
Ch- ilms om malic acid solu ions showed %T alues anged
11.45–4.28%, 29.06–18.15% and 51.34–43.85% in he UVC, UVB and
UVA egions, espec i ely. Simila end was obse ed o Ch- ilms using
lac ic acid as sol en , wi h a e age alues anged be ween 13.75 and
5.53%, 27.13–21.12% and 48.27–41.69%, espec i ely. Pu e chi osan
samples showed simila da a and spec a ha hose p e iously epo ed
[10,42]. Da a e ealed ha he ac i e compounds o TTEO p omp ed a
sligh ly inc ease o he abso bance alues, enhancing he UV-blocking
p ope ies o he o mula ed ilms. No signi ican di e ences we e
ound as unc ion o he sol en used o p epa e he ilm- o ming
solu ion.
These esul s we e ema kable in he UV-C egion, ob aining ans-
mi ance alues below 6%. The ex a p o ec ion o an ioxidan agen s in
Ch- ilms agains oxida i e p ocesses induced by UV ligh we e also
obse ed by he addi ion o min and pomeg ana e peel ex ac [43],
clo e essen ial oil [44] ellagic acid [45], ginge , osema y, sage, ea ee
and hyme essen ial oils [46] o
α
- ocophe ol among o he s samples
[47]. On he o he hand, [46] epo ed lowe blocking capaci y o he
essen ial oils han hei s hyd o-alcoholic ex ac s. Besides he UV-
blocking p ope ies, he de eloped ilms should also be p e y ans-
pa en o achie e he app o al o he consume s. The calcula ed ans-
pa ency alues (Table 4) emained in he ange 52.07–48.80 and
51.10–43.14 o Ch- ilms om malic acid and lac ic acid solu ions,
Table 4
Op ical p ope ies o he chi osan-based ilms.
Film
samples
UV-C UV-B UV-A T anspa ency Opaci y
%T %T %T
CH_M1 11.45 ±2.85 29.06 ±
5.69
51.34 ±
3.63
51.88 3.01
CH_M2 9.86 ±1.97 26.72 ±
4.02
49.28 ±
3.29
48.80 3.29
CH_M3 7.73 ±1.54 23.43 ±
3.29
48.14 ±
2.46
52.07 3.06
CH_M4 4.28 ±2.99 18.15 ±
6.12
43.85 ±
6.72
49.03 3.48
CH_L1 13.75 ±
1.80⋅10
−2
27.13 ±
6.23
48.27 ±
4.90
49.35 2.79
CH_L2 9.99 ±
2.23⋅10
−2
17.34 ±
11.79
41.70 ±
13.67
46.49 2.83
CH_L3 6.30 ±
7.07⋅10
−4
14.55 ±
5.80
38.89 ±
6.23
43.14 2.72
CH_L4 5.53 ±0.46 21.12 ±
6.24
41.69 ±
6.33
51.10 3.62
CH - chi osan; M - malic acid; L - lac ic acid.
UV-C (200–280 nm).
UV-B (280–315 nm).
UV-A (315–400 nm).
%T - pe cen age o ansmi ance.
Values a e exp essed as mean ±s anda d de ia ion (SD).
Di e en le e s in he same column indica e signi ican di e ences (p ˂ 0.05).
Expe imen s a e de ined in Table 1.
Table 5
Colou pa ame e s o he chi osan-based ilms.
Film samples L* a* b*
CH_M1 91.41 ±0.18 −0.58 ±0.13 4.12 ±0.68
CH_M2 90.10 ±0.77 −0.41 ±0.02 4.09 ±0.34
CH_M3 91.63 ±0.04 −0.46 ±0.10 5.38 ±0.43
CH_M4 90.15 ±2.29 −0.34 ±0.16 6.51 ±1.52
CH_L1 91.85 ±0.35 −0.93 ±0.16 5.73 ±1.12
CH_L2 91.49 ±3.59 −1.11 ±0.33 5.64 ±2.38
CH_L3 91.03 ±0.17 −1.27 ±0.27 6.84 ±1.87
CH_L4 90.16 ±1.17 −0.80 ±0.04 7.30 ±1.70
CH - chi osan; M - malic acid; L - lac ic acid.
L*, ligh ness: black =0 and whi e =100; a*, g een = − a* and ed = +a*); b*,
blue = − b* and yellow = +b*.
Values a e exp essed as mean ±s anda d de ia ion (SD).
Expe imen s a e de ined in Table 1.
P. Caz´
on e al.
In e na ional Jou nal o Biological Mac omolecules 186 (2021) 365–376
373
Fig. 5. The mog a ime y and di e en ial scanning calo ime y o he selec ed chi osan - ea ee essen ial oil ilms.
P. Caz´
on e al.
