Philippine e-Jou nal o Applied Resea ch and De elopmen 15(2025), 51-61 ISSN 2449-3694 (Online)
PURL: h ps://peja d.slu.edu.ph/ ol.15/2025.11.21.pd h ps://doi.o g/10.5281/zenodo.17694788
Physicochemical p ope ies and an ibac e ial ac i i y o co ns a ch-
based ilms inco po a ed wi h lemong ass (Cymbopogon ci a us)
essen ial oil
Be ina Dannah M. Villanue a1, Adeliza A. Do ado1*, Ma. Josie V. Sumague1, and Romel M.
Felismino1
1Ins i u e o Food Science and Technology, College o Ag icul u e and Food Science, Uni e si y o he Philippine
Los Baños
*[email p o ec ed]
Recei ed, 02 May 2025; Accep ed, 13 No embe 2025; Published, 21 No embe 2025
Copy igh @ 2025 B.D.M. Villanue a, A.A. Do ado, MJ.V. Sumague, and R.M. Felismino. This is an open access
a icle dis ibu ed unde he C ea i e Commons A ibu ion License, which pe mi s un es ic ed use, dis ibu ion,
and ep oduc ion in any medium, p o ided he o iginal wo k is p ope ly ci ed.
Abs ac
Bioplas ic ilms p epa ed om co ns a ch inco po a ed wi h lemong ass essen ial oil (LEO)
we e e alua ed in e ms o hei physicochemical and an ibac e ial p ope ies. The ilms we e
p epa ed ia solu ion cas ing o con ain di e en amoun s o LEO (0%, 0.5%, 1%, and 2% w/w).
The ilms we e cha ac e ized o hickness, colo p ope ies, mois u e con en , ensile s eng h,
and elonga ion a b eak. An ibac e ial ac i i y agains S aphylococcus au eus (BIOTECH 1582)
and Salmonella yphimu ium (BIOTECH 1826) was de e mined ia disk di usion using 6 mm
ilm discs on Muelle –Hin on aga . Signi ican imp o emen in elonga ion a b eak (25.06 ±
7.39%) a 0.5% LEO, and inc ease in ilm hickness and mois u e con en wi h LEO addi ion
we e obse ed. All ilms con aining LEO displayed s ong an ibac e ial ac i i y. In e es ingly,
he con ol ilm (0% LEO) exhibi ed no able baseline an ibac e ial ac i i y (inhibi ion zones
>29 mm) p obably due o he inega and glyce ol in i s o mula ion. Conside able inc ease
in an imic obial ac i i y occu s wi h 2% LEO agains S. au eus. O e all, ilms p epa ed wi h
0.5% LEO showed he bes possible balance be ween mechanical lexibili y and an imic obial
ac i i y, sugges ing ha his o mula ion may be used in p epa ing ac i e ood packaging
ma e ials.
Keywo ds: an ibac e ial packaging, elonga ion a b eak, mic obial zone o inhibi ion, ensile
s eng h
Philippine e-Jou nal o Applied Resea ch and De elopmen
Websi e: peja d.slu.edu.ph ISSN 2449-3694 (Online)
In oduc ion
Food packaging plays a c ucial ole as i
p o ec s ood ing edien s and p oduc s om
po en ial damage and de e io a ion (Pe koska e
al., 2021; Shanbhag e al., 2023). Con en ional
ood packaging includes pape , glass, plas ics,
and me als. Al hough o e 20% o pape and
pape boa d a e ecycled, a ious plas ics a e
ecycled a a e y low a e (< 20%) (Jee ahan
& Chand aseka an, 2019). Thus, he common
p ac ice o disca ding packaging ma e ials
especially con en ional plas ics poses a se ious
en i onmen al p oblem, as hese a e widely
used and a e non-biodeg adable. Mo eo e ,
con en ional plas ics a e de i ed om non-
enewable pe oleum ese es and gene a e
ca bon dioxide and oxic subs ances when
incine a ed (Pe koska e al., 2021).
Due o p essing issues su ounding he use
o con en ional plas ics, he ood indus y now
conside s he de elopmen o biodeg adable,
ac i e, and edible packaging ma e ials among
i s op p io i ies. These al e na i e packaging
G.G.L.-Co Yu Kang, T.J. Pa eñas, and N.P. Nolasco
52 B.D.M. Villanue a, A.A. Do ado, MJ.V. Sumague, and R.M. Felismino
ma e ials include ilms ha a e p epa ed om
edible ma e ials such as lipids, p o eins, and
polysaccha ides. These bioplas ic ilms may be
o med in o w aps, pouches, bags, capsules, and
casings (Jee ahan & Chand aseka an, 2019).
In ecen yea s, s a ch-based ilms ha e
a ac ed conside able a en ion due o hei
inhe en biodeg adabili y, high a ailabili y, and
low cos (Do ado e al., 2017). S a ch is a na u al
polyme ha may be o med in o colo less, odo less
ilms wi h low oxygen pe meabili y (Cano e al.,
2014; Ga cia e al., 2020). Howe e , s a ch-based
ilms su e om high wa e apo pe meabili y
because o he abundance o hyd oxyl g oups
in s a ch, and likewise exhibi poo mechanical
p ope ies (Do ado e al., 2017; Ga cia e al.,
2020). To o e come hese d awbacks, a plas icize
is added o he ilm ma ix o imp o e lexibili y
and mechanical p ope ies (Yousse & El-Sayed,
2018). Low-molecula weigh polyols a e o en
used as plas icize s, wi h glyce ol being he mos
s udied polyol. Molecula weigh is associa ed
wi h he e icacy o a plas icize : he smalle
he molecule, he g ea e he plas icizing e ec .
Aside om plas icize s, c osslinking agen s such
as inega can be added o imp o e he ilm’s
s eng h. Vinega is an ace ic acid solu ion ha
eleases ace a e and hyd ogen ions ha , in u n,
eac wi h s a ch polyme s so ha hey become
diso de ed. This diso de a ising om he
ioniza ion o wa e and ace ic acid ende s he
ilm mo e homogenous (Shanbhag e al., 2023).
To ob ain unique unc ionali y, addi i es
such as an imic obial agen s, colo s, and
la o s a e inco po a ed in o he s a ch ma ix
depending on i s in ended use (Bilal, Zhao &
Iqbal, 2020). Fo example, o p e en mic obial
g ow h, s a ch-based ilms can be in used wi h
an imic obial agen s such as essen ial oils (EOs).
When inco po a ed in o he ilm ma ix, EOs can
be eleased in o he ood upon con ac wi h he
ilm su ace (do E angelo, 2019). Fu he mo e,
EOs se e o educe he wa e apo pe meabili y
o s a ch-based ilms (Ga cia e al., 2020). EOs
ha e an oily and ola ile na u e which in luence
he s uc u al cha ac e is ics and deg ee o
hyd ophobici y o polyme ic ilms, hus al e ing
hei mechanical and ba ie p ope ies (do
E angelo e al., 2019; A a es & Chi al , 2016).
EOs a e he bes eplacemen o pe oleum-
de i ed addi i es in ood packaging ma e ials
because hey a e na u ally abundan , eco-
iendly, and possess supe io an imic obial
and an ioxidan a ibu es (Zubai e al., 2022).
Howe e , al hough he an imic obial p ope ies
o se e al EOs in biodeg adable ilms ha e been
widely s udied, he e a e e y ew epo s on
hei e ec s on ilm p ope ies (do E angelo e
al., 2019). Some such epo s include wo ks on
s a ch/chi osan ilms in used wi h lemong ass
essen ial oil (LEO) whe ein an imic obial
ac i i y was e alua ed along wi h wa e apo
pe meabili y, wa e solubili y, and mechanical
p ope ies (Pe dana e al. 2021); on cassa a
s a ch/chi osan ilms inco po a ed wi h LEO
whe e an imic obial ac i i y and mechanical
p ope ies we e e alua ed agains s o age ime
(Pe dana e al., 2022); and, on co ns a ch ilms
con aining Zan hoxylum bungeanum essen ial
oil (ZYO) whe ein an imic obial ac i i y and
physical cha ac e is ics (mo phology, op ics,
mechanical and ba ie p ope ies) we e epo ed
(Wang e al., 2020). In his s udy, we will epo
a combina ion o mic obiological (e icacy agains
G am-posi i e and G am-nega i e bac e ia) and
physicochemical (su ace colo , anspa ency,
mois u e con en , ensile s eng h, elonga ion)
e alua ions o LEO-in used ilms p epa ed om
co ns a ch, glyce ol, and inega , wi h ocus on
he e ec s o a ying LEO concen a ions (0.5–
2% w/w LEO). As such, his s udy cen e s on he
po en ial o co ns a ch ma ices combined wi h
commonly a ailable plas icize and acid o help
add ess he dual challenge o en i onmen al
pollu ion om syn he ic packaging and ood
spoilage due o mic obial con amina ion.
Fu he mo e, his s udy p o ides insigh s in o
op imizing ilm composi ion o mechanical
pe o mance and an imic obial e icacy.
Me hodology
P epa a ion o ilms
Comme cial co ns a ch (C eam pu e
co ns a ch), dis illed wa e (Absolu e), and 5%
acidi y inega (Heinz dis illed inega ) we e
pu chased om a local supe ma ke . Labo a o y-
g ade glyce ol (Scha lau A.R.) and LEO we e
p ocu ed om Chemline Scien i ic Co po a ion
and Young Li ing Philippines, espec i ely.
53
Philippine e-Jou nal o Applied Resea ch and De elopmen
Websi e: peja d.slu.edu.ph ISSN 2449-3694 (Online)
Philippine e-Jou nal o Applied Resea ch and De elopmen 15(2025), 51-61
The co ns a ch-based ilms we e p epa ed
ia solu ion cas ing acco ding o he me hods
o Resianing um e al. (2016), do E angelo e
al. (2019) and Wang e al. (2020) wi h some
modi ica ions. The s a ch-based mix u e
consis ed o dis illed wa e , co ns a ch, inega ,
and glyce ol o which LEO was added a di e en
amoun s. Films we e p epa ed by mixing
co ns a ch (7.67% w/w), glyce ol (7.67% w/w),
and inega (7.67% w/w) in wa e (77% w/w) and
hea ed a 80–85 °C using a co e ed double-boile
s eame o 15 min o p e en ola iliza ion o
oil. LEO was added di ec ly o he solu ion a 0%,
0.5%, 1%, and 2% w/w o o al solu ion weigh
(Table 1) and s i ed ho oughly. The mix u e
(Figu e 1a) was cas on o wax-pape -lined ays
(380 × 280 mm) and d ied a 40 °C o 72 h. D ied
ilms we e s o ed in desicca o s un il analysis.
The p epa a ion o packaging ilms was done in
iplica es o each ea men .
Film hickness
The hickness o he ilms was measu ed
as he a e age o en measu emen s aken a
he cen e and pe iphe y o he ilm using a
mic ome e calipe .
Tensile s eng h and elonga ion a b eak
The ensile s eng h (TS) and pe cen
elonga ion a b eak (%E) o he shee s we e
de e mined acco ding o he ASTM D882
(S anda d Tes Me hod o Tensile P ope ies
o Thin Plas ic Shee ing) using an Ins on
Uni e sal Tes ing Machine (Model 5585H). All
Table 1
Fo mula ion o he co ns a ch-based ilms.
Componen s % Lemong ass essen ial oil (LEO)
0 0.5 1.0 2.0
Co ns a ch (g) 10 10 10 10
Dis illed wa e (g) 100 99.35 98.7 97.4
Glyce ol (g) 10 10 10 10
Vinega (g) 10 10 10 10
LEO (g) 0 0.65 1.3 2.6
To al (g) 130 130 130 130
ilm samples we e p e-condi ioned a 23 ± 2°C and
50 ± 5% ela i e humidi y o a leas 48 h p io o
mechanical and colo es ing in acco dance wi h
ASTM D618 (S anda d P ac ice o Condi ioning
Plas ics o Tes ing). Fo e e y o mula ion, en
eplica es we e es ed. To accoun o ba ch- o-
ba ch a ia ion, hese eplica es we e aken om
each o he h ee independen ly p epa ed ilm
shee s o each ea men . The TS was calcula ed
using Eq. 1 whe e Lp is he peak load (N) and a
is he c oss-sec ional a ea o he s ips (m2) while
he %E was de e mined using Eq. 2 whe e ∆l is
he change in leng h a b eaking poin (mm) and
l is he o iginal leng h (mm) o he ilm s ip.
3
g ade glyce ol (Scha lau A.R.) and LEO we e p ocu ed om Chemline Scien i ic Co po a ion 91
and Young Li ing Philippines, espec i ely. 92
The co ns a ch-based ilms we e p epa ed ia solu ion cas ing acco ding o he me hods 93
o Resianing um e al. (2016), do E angelo e al. (2019) and Wang e al. (2020) wi h some 94
modi ica ions. The s a ch-based mix u e consis ed o dis illed wa e , co ns a ch, inega , and 95
glyce ol o which LEO was added a di e en amoun s. Films we e p epa ed by mixing 96
co ns a ch (7.67% w/w), glyce ol (7.67% w/w), and inega (7.67% w/w) in wa e (77% w/w) 97
and hea ed a 80–85 °C using a co e ed double-boile s eame o 15 min o p e en 98
ola iliza ion o oil. LEO was added di ec ly o he solu ion a 0%, 0.5%, 1%, and 2% w/w o 99
o al solu ion weigh (Table 1) and s i ed ho oughly. The mix u e (Figu e 1a) was cas on o 100
wax-pape -lined ays (380 × 280 mm) and d ied a 40 °C o 72 h. D ied ilms we e s o ed in 101
desicca o s un il analysis. The p epa a ion o packaging ilms was done in iplica es o each 102
ea men . 103
Table 1. Fo mula ion o he co ns a ch-based ilms. 104
Componen s
% Lemong ass essen ial oil (LEO)
0
0.5
1.0
2.0
Co ns a ch (g)
10
10
10
10
Dis illed wa e (g)
100
99.35
98.7
97.4
Glyce ol (g)
10
10
10
10
Vinega (g)
10
10
10
10
LEO (g)
0
0.65
1.3
2.6
To al (g)
130
130
130
130
105
106
Film hickness 107
108
The hickness o he ilms was measu ed as he a e age o en measu emen s aken a 109
he cen e and pe iphe y o he ilm using a mic ome e calipe . 110
111
Tensile s eng h and elonga ion a b eak 112
113
The ensile s eng h (TS) and pe cen elonga ion a b eak (%E) o he shee s we e 114
de e mined acco ding o he ASTM D882 (S anda d Tes Me hod o Tensile P ope ies o Thin 115
Plas ic Shee ing) using an Ins on Uni e sal Tes ing Machine (Model 5585H). All ilm samples 116
we e p e-condi ioned a 23 ± 2°C and 50 ± 5% ela i e humidi y o a leas 48 h p io o 117
mechanical and colo es ing in acco dance wi h ASTM D618 (S anda d P ac ice o 118
Condi ioning Plas ics o Tes ing). Fo e e y o mula ion, en eplica es we e es ed. To 119
accoun o ba ch- o-ba ch a ia ion, hese eplica es we e aken om each o he h ee 120
independen ly p epa ed ilm shee s o each ea men . The TS was calcula ed using Eq. 1 121
whe e 𝐿𝐿𝐿𝐿𝑝𝑝𝑝𝑝 is he peak load (N) and 𝑎𝑎𝑎𝑎 is he c oss-sec ional a ea o he s ips (m2) while he %E 122
was de e mined using Eq. 2 whe e ∆𝑙𝑙𝑙𝑙 is he change in leng h a b eaking poin (mm) and 𝑙𝑙𝑙𝑙 is 123
he o iginal leng h (mm) o he ilm s ip. 124
𝑇𝑇𝑇𝑇𝑇𝑇𝑇𝑇 =
𝐿𝐿𝐿𝐿
𝑝𝑝𝑝𝑝
𝑎𝑎𝑎𝑎
Eq. 1
%𝐸𝐸𝐸𝐸= �
∆𝑙𝑙𝑙𝑙
𝑙𝑙𝑙𝑙
� × 100
Eq. 2
Eq. 1
Eq. 2
Colo p ope ies
The colo o he co ns a ch-based ilms was
measu ed using he CIELAB coo dina es (L*,
a*, and b*) ob ained om Ch oma Me e CR-400
(Minol a, Tokyo, Japan). P io o measu emen s,
he ins umen was calib a ed using he
con en ional wo-poin me hod – ze o calib a ion
(black s anda d) and whi e calib a ion using
he whi e calib a ion ile p o ided by he
manu ac u e . Films om each o mula ion was
es ed in h ee uns, wi h h ee eplica es o
54 B.D.M. Villanue a, A.A. Do ado, MJ.V. Sumague, and R.M. Felismino
each un. The whi eness index (WI) ha ac s
as an indi ec indica o o changes in he isual
cha ac e is ics o he ilm (Ga cia e al., 2020;
Wang e al., 2020) was compu ed using Eq. 3.
4
Colo p ope ies 125
The colo o he co ns a ch-based ilms was measu ed using he CIELAB coo dina es 126
(L*, a*, and b*) ob ained om Ch oma Me e CR-400 (Minol a, Tokyo, Japan). P io o 127
measu emen s, he ins umen was calib a ed using he con en ional wo-poin me hod – ze o 128
calib a ion (black s anda d) and whi e calib a ion using he whi e calib a ion ile p o ided by 129
he manu ac u e . Films om each o mula ion was es ed in h ee uns, wi h h ee eplica es 130
o each un. The whi eness index (WI) ha ac s as an indi ec indica o o changes in he isual 131
cha ac e is ics o he ilm (Ga cia e al., 2020; Wang e al., 2020) was compu ed using Eq. 3. 132
𝑊𝑊𝑊𝑊𝑊𝑊𝑊𝑊 =100 −�(100 −𝐿𝐿𝐿𝐿
∗
) + 𝑎𝑎𝑎𝑎
∗2
+𝑏𝑏𝑏𝑏
∗2
Eq. 3
133
Mois u e Con en 134
135
The mois u e con en o he ilms was ob ained acco ding o he me hods o Wang e 136
al. (2020) wi h some modi ica ions. The samples we e i s condi ioned a 60 ± 5% RH o 48 137
h. App oxima ely 1 g o a ilm sample was cu in o iny pieces and placed in a a ed c ucible. 138
The c ucible con aining he sample was placed in an o en a 100 ± 5 oC o 24 h, and hen 139
allowed o equilib a e a oom empe a u e in a desicca o be o e e-weighing. The ea e , he 140
c ucible was e u ned o he o en o 30 min, and hen cooled and eweighed. These e-d ying 141
and eweighing s eps we e epea ed un il he weigh o he wo p e ious eadings did no di e 142
by mo e han 0.001 g. The mois u e con en (MC) o he ilm was calcula ed using Eq. 4. 143
𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀 =
𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐 𝑤𝑤𝑤𝑤𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑖𝑖𝑖𝑖ℎ𝑐𝑐𝑐𝑐−𝑓𝑓𝑓𝑓𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑎𝑎𝑎𝑎𝑓𝑓𝑓𝑓 𝑤𝑤𝑤𝑤𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑖𝑖𝑖𝑖ℎ𝑐𝑐𝑐𝑐
𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐 𝑤𝑤𝑤𝑤𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑖𝑖𝑖𝑖ℎ𝑐𝑐𝑐𝑐
× 100 Eq. 4
144
Mic obial ac i i y o he ilms 145
146
P io o he conduc o he mic obial ac i i y es , G am-posi i e S aphylococcus au eus 147
(BIOTECH 1582) and G am-nega i e Salmonella yphimu ium (BIOTECH 1826) ob ained 148
om he Uni e si y o he Philippines Los Baños - Na ional Ins i u e o Biology and 149
Bio echnology (UPLB-BIOTECH), we e g own a oom empe a u e gene a ing 9.63 × 108 150
CFU mL-1 and 3.3 × 109 CFU mL-1 o S. au eus and S. yphimu ium, espec i ely. 151
The an imic obial ac i i y o he ilms we e e alua ed by disk di usion assay acco ding 152
o he Ki by-Baue me hod (Ch is enson e al., 2017) wi h modi ica ions. The ilms we e cu 153
in o 6-mm diame e discs and placed on he su ace o he Muelle -Hin on aga (MHA) 154
p e iously inocula ed wi h 0.1 mL o he bac e ia cul u e h ough sp ead pla ing (Wang e al., 155
2020). The u bidi y o he inoculan was adjus ed wi h s e ile b o h p io o being sp ead on o 156
he aga pla es o an app oxima e cell densi y o 1.5 × 108 CFU mL-1. The an imic obial 157
e ec s o co ns a ch-based ilms con aining di e en concen a ions o LEO agains S. au eus 158
and S. yphimu ium we e ca ied ou using a zone o inhibi ion assay on MHA. The pla es we e 159
hen incuba ed a 37 °C o 24 h. A e incuba ion, he inhibi ion zone was measu ed on he 160
Eq. 3
Mois u e Con en
The mois u e con en o he ilms was
ob ained acco ding o he me hods o Wang e
al. (2020) wi h some modi ica ions. The samples
we e i s condi ioned a 60 ± 5% RH o 48 h.
App oxima ely 1 g o a ilm sample was cu in o
iny pieces and placed in a a ed c ucible. The
c ucible con aining he sample was placed in an
o en a 100 ± 5 oC o 24 h, and hen allowed o
equilib a e a oom empe a u e in a desicca o
be o e e-weighing. The ea e , he c ucible was
e u ned o he o en o 30 min, and hen cooled
and eweighed. These e-d ying and eweighing
s eps we e epea ed un il he weigh o he wo
p e ious eadings did no di e by mo e han
0.001 g. The mois u e con en (MC) o he ilm
was calcula ed using Eq. 4.
4
Colo p ope ies 125
The colo o he co ns a ch-based ilms was measu ed using he CIELAB coo dina es 126
(L*, a*, and b*) ob ained om Ch oma Me e CR-400 (Minol a, Tokyo, Japan). P io o 127
measu emen s, he ins umen was calib a ed using he con en ional wo-poin me hod – ze o 128
calib a ion (black s anda d) and whi e calib a ion using he whi e calib a ion ile p o ided by 129
he manu ac u e . Films om each o mula ion was es ed in h ee uns, wi h h ee eplica es 130
o each un. The whi eness index (WI) ha ac s as an indi ec indica o o changes in he isual 131
cha ac e is ics o he ilm (Ga cia e al., 2020; Wang e al., 2020) was compu ed using Eq. 3. 132
𝑊𝑊𝑊𝑊𝑊𝑊𝑊𝑊 =100 −�(100 −𝐿𝐿𝐿𝐿
∗
) + 𝑎𝑎𝑎𝑎
∗2
+𝑏𝑏𝑏𝑏
∗2
Eq. 3
133
Mois u e Con en 134
135
The mois u e con en o he ilms was ob ained acco ding o he me hods o Wang e 136
al. (2020) wi h some modi ica ions. The samples we e i s condi ioned a 60 ± 5% RH o 48 137
h. App oxima ely 1 g o a ilm sample was cu in o iny pieces and placed in a a ed c ucible. 138
The c ucible con aining he sample was placed in an o en a 100 ± 5 oC o 24 h, and hen 139
allowed o equilib a e a oom empe a u e in a desicca o be o e e-weighing. The ea e , he 140
c ucible was e u ned o he o en o 30 min, and hen cooled and eweighed. These e-d ying 141
and eweighing s eps we e epea ed un il he weigh o he wo p e ious eadings did no di e 142
by mo e han 0.001 g. The mois u e con en (MC) o he ilm was calcula ed using Eq. 4. 143
𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀 =
𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐 𝑤𝑤𝑤𝑤𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑖𝑖𝑖𝑖ℎ𝑐𝑐𝑐𝑐−𝑓𝑓𝑓𝑓𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑎𝑎𝑎𝑎𝑓𝑓𝑓𝑓 𝑤𝑤𝑤𝑤𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑖𝑖𝑖𝑖ℎ𝑐𝑐𝑐𝑐
𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐 𝑤𝑤𝑤𝑤𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑖𝑖𝑖𝑖ℎ𝑐𝑐𝑐𝑐
× 100
Eq. 4
144
Mic obial ac i i y o he ilms 145
146
P io o he conduc o he mic obial ac i i y es , G am-posi i e S aphylococcus au eus 147
(BIOTECH 1582) and G am-nega i e Salmonella yphimu ium (BIOTECH 1826) ob ained 148
om he Uni e si y o he Philippines Los Baños - Na ional Ins i u e o Biology and 149
Bio echnology (UPLB-BIOTECH), we e g own a oom empe a u e gene a ing 9.63 × 108 150
CFU mL-1 and 3.3 × 109 CFU mL-1 o S. au eus and S. yphimu ium, espec i ely. 151
The an imic obial ac i i y o he ilms we e e alua ed by disk di usion assay acco ding 152
o he Ki by-Baue me hod (Ch is enson e al., 2017) wi h modi ica ions. The ilms we e cu 153
in o 6-mm diame e discs and placed on he su ace o he Muelle -Hin on aga (MHA) 154
p e iously inocula ed wi h 0.1 mL o he bac e ia cul u e h ough sp ead pla ing (Wang e al., 155
2020). The u bidi y o he inoculan was adjus ed wi h s e ile b o h p io o being sp ead on o 156
he aga pla es o an app oxima e cell densi y o 1.5 × 108 CFU mL-1. The an imic obial 157
e ec s o co ns a ch-based ilms con aining di e en concen a ions o LEO agains S. au eus 158
and S. yphimu ium we e ca ied ou using a zone o inhibi ion assay on MHA. The pla es we e 159
hen incuba ed a 37 °C o 24 h. A e incuba ion, he inhibi ion zone was measu ed on he 160
Eg. 4
Mic obial ac i i y o he ilms
P io o he conduc o he mic obial
ac i i y es , G am-posi i e S aphylococcus
au eus (BIOTECH 1582) and G am-nega i e
Salmonella yphimu ium (BIOTECH 1826)
ob ained om he Uni e si y o he Philippines
Los Baños - Na ional Ins i u e o Biology and
Bio echnology (UPLB-BIOTECH), we e g own
a oom empe a u e o a ound 30oC gene a ing
9.63 x 108 CFU mL-1 and 3.3 x 109 CFU mL-1 o S.
au eus and S. yphimu ium, espec i ely.
The an imic obial ac i i y o he ilms we e
e alua ed by disk di usion assay acco ding o
he Ki by-Baue me hod (Ch is enson e al.,
2017) wi h modi ica ions. The ilms we e cu in o
6-mm diame e discs and placed on he su ace
o he Muelle -Hin on aga (MHA) p e iously
inocula ed wi h 0.1 mL o he bac e ia cul u e
h ough sp ead pla ing (Wang e al., 2020). The
u bidi y o he inoculan was adjus ed wi h
s e ile b o h p io o being sp ead on o he aga
pla es o an app oxima e cell densi y o 1.5 x 108
CFU mL-1. The an imic obial e ec s o co ns a ch-
based ilms con aining di e en concen a ions
o LEO agains S. au eus and S. yphimu ium
we e ca ied ou using a zone o inhibi ion assay
on MHA. The pla es we e hen incuba ed a 37
°C o 24 h. A e incuba ion, he inhibi ion zone
was measu ed on he ilm disks (Manab e al.,
2011). The ilm wi h no EO (0% LEO) se ed as
he un ea ed con ol. Th ee eplica es om each
o he h ee uns o each ea men we e es ed.
S a is ical Analysis
All means and s anda d de ia ions we e
calcula ed and analyzed s a is ically using
Mic oso Excel. Signi ican di e ences
be ween he samples we e de e mined o all
cha ac e is ics measu ed using analysis o
a iance (ANOVA) a a 95% con idence le el and
Tukey pai wise compa ison.
Resul s
All ilms we e mac oscopically homogeneous
wi hou isible oil d ople s o exuda es. Figu e
1a shows a sample o hea ed ilm solu ion on ay
while Figu e 1b shows samples o d ied ilms.
Tables 2 and 3 lis he physiochemical
cha ac e is ics o he ilms. Films wi h 0.5%
LEO exhibi ed he highes ensile s eng h
(0.966 ± 0.252 MPa) and elonga ion a b eak
(25.06 ± 7.39%), ou pe o ming bo h un ea ed
ilms and ilms ea ed wi h highe amoun s o
LEO. I appea s ha dis up ion o he polyme
ne wo k occu ed a highe amoun s o LEO,
esul ing in weake mechanical pe o mance.
Loss o anspa ency wi h inco po a ion o
LEO sugges s i s en apmen in mic od ople s,
consis en wi h li e a u e epo s on EO–polyme
ilms. Also, inco po a ion o LEO signi ican ly
inc eased ilm hickness, consis en wi h
li e a u e epo s.
The ilms exhibi an ibac e ial ac i i y
as indica ed by he p esence o ci cula zones
o inhibi ion in disk di usion assays o S.
55
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Table 2
Thickness, ensile s eng h, and elonga ion a b eak o co ns a ch-based ilms inco po a ed wi h
lemong ass essen ial oil (LEO).
T ea men
(% LEO)
Thickness (mm)
Tensile S eng h (MPa) Pe cen age
Elonga ion a B eak
0% (Con ol) 0.120 ± 0.034b 0.860 ± 0.303ab 14.072 ± 3.101b
0.5% 0.177 ± 0.051a0.966 ± 0.252a25.060 ± 7.390a
1% 0.161 ± 0.041ab 0.594 ± 0.171bc 14.500 ± 2.866b
2% 0.186 ±0.029a0.475 ±0.166c12.930 ±3.880b
Each da a poin ep esen s he mean ± s anda d de ia ion o 10 eplica es ob ained om each o mula ion. The mul iple com-
pa isons we e de e mined by Tukey a p < 0.05. Means in he same column wi h di e en le e s a e signi ican ly di e en .
Table 3
Colo alues, whi eness index(WI), and mois u e con en o co ns a ch-based ilms inco po a ed wi h
lemong ass essen ial oil (LEO).
T ea men
(% LEO) L* a* b* WI Mois u e
Con en (%)
0% (Con ol)
92.249±
0.610 a
-0.049 ± 0.043a3.690 ± 0.218b91.412 ± 0.597a22.195 ± 0.331c
0.5% 91.456 ±
0.490a
-0.303 ± 0.068b4.131 ± 0.311b90.496 ± 0.409a24.920 ± 0.364b
1% 91.949 ±
0.963a
-0.421 ± 0.086c4.950 ± 0.566a90.531 ± 1.040a28.839 ± 0.778a
2% 92.082 ±
1.253a
-0.410 ± 0.103c4.919 ± 0.530a90.661 ± 1.297a24.145 ± 0.634b
Each da a poin ep esen s he mean ± s anda d de ia ion o iplica es ob ained om iplica e p epa a ions o ilms. The
mul iple compa isons we e de e mined by Tukey a p < 0.05. Means in he same column wi h di e en le e s a e signi ican ly
di e en . L*, a*, b* a e he coo dina es o he CIELAB colo space diag am. L* (0 o 50 is conside ed da k while 51 o 100 is
conside ed ligh ), a* (posi i e alues indica e edness while nega i e alues indica e g eenness), b* (posi i e alues indica e
yellowness while nega i e alues indica e blueness).
Figu e 1
(a) a ay o ilm solu ion, and (b) samples o d ied LEO- ea ed co ns a ch ilms.
56 B.D.M. Villanue a, A.A. Do ado, MJ.V. Sumague, and R.M. Felismino
yphimu ium (Figu e 2a) and S. au eus (Figu e
2b). The diame e o hese zones (Figu e 2c)
was measu ed and summa ized in Table 4.
While Tukey’s HSD es iden i ies di e ences
be ween ea men s, he biological signi icance
was de e mined based on he classi ica ion
o inhibi ion zone diame e s. All samples
demons a ed e y s ong inhibi ion (>20 mm),
indica ing e ec i e an ibac e ial ac i i y.
Figu e 2
(a) disk di usion assays o S. yphimu ium (L-R: 0%, 0.5%, 1%, 2%), (b) S. au eus (L-R: 0%, 0.5%, 1%,
2%), and (c) he me hod o es ima ing diame e s o mic obial inhibi ion zones.
Table 4
Zone o inhibi ion o an ibac e ial ac i i y o co ns a ch-based ilms inco po a ed wi h lemong ass
essen ial oil (LEO).
T ea men (% LEO) Diame e o Zone o Inhibi ion (mm)
S. au eus S. yphimu ium
0% (Con ol) 32.78±4.60 ab 29.33±1.94 bc
0.5% 32.22±2.91 abc 28.44±1.24 c
1% 31.89±3.18 abc 28.33±2.50 c
2% 34.22±1.72a 30.67±1.80 abc
Each da a poin ep esen s he mean ± s anda d de ia ion o iplica es ob ained om iplica e p epa a ions o ilms. The mul-
iple compa isons we e de e mined by Tukey a p < 0.05. Means ha do no sha e a le e a e signi ican ly di e en .
Inhibi ion zone diame e s ≥20 mm a e indica i e o e y s ong an ibac e ial ac i i y (Da is and S ou , 1971).
57
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Discussion
Thickness o ilms
Inco po a ing LEO signi ican ly inc eased
he hickness o he co ns a ch-based ilms (Table
2). This esul is in ag eemen wi h indings
om s a ch-based ilms inco po a ed wi h
Zan hoxylum bungeanum essen ial oil (Wang e
al., 2020), co ns a ch ilms wi h o ange essen ial
oil (do E angelho e al., 2019), ca boxyme hyl
xylan ilms wi h lico ice essen ial oil (Luis e
al., 2019), and cassa a s a ch-based edible ilms
wi h lemong ass oil (Resianing um e al., 2016).
In hei s udy, Luis e al. (2019) a ibu ed his
beha io o he en apmen o mic od ople s o
lico ice essen ial oil in o he ilm ma ix, which
gi es ise o less dense and hicke ilms.
In his s udy, he ilm wi h he highes
amoun o LEO (2% w/w) showed he highes
mean hickness. Howe e , he e ec s o
a ying amoun s o LEO on he hickness o
he ilms a e no signi ican ly di e en om
each o he . Meanwhile, al hough he hickness
o ilms p epa ed wi h 0%, 0.5% and 2% LEO
a e signi ican ly di e en , he hickness o
ilms p epa ed wi h 0% and 1% LEO a e no
signi ican ly di e en . Ne e heless, he ideal
hickness o ilms which is < 0.25 mm (Ha mi e
al., 2020) was achie ed in his s udy.
In he de elopmen o packaging ma e ials,
he hickness o he ilm is measu ed o he
e alua ion o o he p ope ies – s uc u al,
mechanical, he mal, and ba ie p ope ies. Fo
example, in he case o ba ie p ope ies, hicke
ilms a e desi ed since his would mean a longe
pa h o wa e apo o a e se o pene a e he
ilm.
Tensile s eng h (TS)
The mechanical p ope ies o he ilms we e
signi ican ly in luenced by he addi ion o LEO
(Table 2). The mean TS o he ilm wi h 0.5% LEO
was he highes (0.966 ± 0.252 MPa); howe e , i
did no di e s a is ically om ha o he con ol
ilm (0.860 ± 0.303 MPa). In hei s udy on
cassa a s a ch-based edible ilms, Resianing um
e al. (2016) explained ha LEO con ains solid
componen s ha lessen he in e cellula gaps
in edible ilms which esul s in mo e compac
and i me ilm ma ices. This may explain he
obse ed inc ease in TS in ilms p epa ed wi h
0.5% LEO.
A highe amoun s o LEO (1% and 2%), he
TS dec eased signi ican ly ela i e o he con ol.
Simila esul s we e ob ained o co ns a ch ilms
con aining o ange essen ial oil (do E angelho e
al., 2019); apioca-based edible ilms inco po a ed
wi h cinnamon essen ial oil (U ami e al.,
2019); and chi osan-based ilms inco po a ed
wi h hyme, clo e, and cinnamon essen ial oils
(Hossenei e al., 2009). The dec ease in TS can be
a ibu ed o essen ial oil causing he o ma ion
o a he e ogeneous ilm s uc u e (U ami e al.,
2019) o he b eakup o he polyme ilm ne wo k
(Hossenei e al., 2009). Howe e , o he ac o s
mus be conside ed when e alua ing he TS o
ilms. The concen a ion and composi ion o he
s a ch (i.e., amylose and amylopec in) g ea ly
a ec he ensile s eng h (Ha mi e al., 2020).
Elonga ion a b eak
Compa ed wi h he o he o mula ions,
he ilm p epa ed wi h 0.5% LEO showed a
signi ican ly imp o ed elonga ion a b eak (25.060
± 7.390%), which means ha a plas icizing e ec
occu s a his o mula ion (Table 2), simila
o obse a ions in ilms wi h low amoun s o
essen ial oil (do E angelho e al., 2019). Plas icize
molecules dis up he s a ch’s cohesi eness,
dec ease in e molecula connec ions, and
inc ease polyme mobili y (Resianing um e al.,
2016). Meanwhile, he dec ease in elonga ion a
b eak a highe amoun s o LEO was simila ly
obse ed in he s udy o Hossenei e al. (2009),
whe ein bo h he TS and elonga ion a b eak
dec eased in 1% hyme essen ial oil and 1.5%
clo e essen ial oil o mula ions o chi osan-based
ilms; and in he s udy o U ami e al. (2019)
whe ein bo h he TS and elonga ion a b eak
dec eased when 1% o cinnamon essen ial oil was
added in apioca-based edible ilms. Acco ding o
U ami e al. (2019), essen ial oils c ea e compac
ilm s uc u es, he eby imp o ing con inui y
in polysaccha ide ne wo ks, which leads o a
dec ease in elonga ion. Mo eo e , Hosseini e al.
(2009) explains ha a c oss-linking e ec can be
induced by s ong in e ac ions be ween polyme s
and essen ial oils ha educes he ee olume
and molecula mobili y o polyme s, leading o a
58 B.D.M. Villanue a, A.A. Do ado, MJ.V. Sumague, and R.M. Felismino
dec ease in elonga ion a b eak.
In his s udy, i may be p oposed ha
maximum polyme (i.e., s a ch) mobili y occu s
a 0.5% LEO inco po a ion, which esul ed in a
maximum ilm elonga ion o abou 25%. Films
made om polysaccha ides ha e elonga ion
alues om 1 o 80% (Ha mi e al., 2020), which
pu s he elonga ion a b eak o he LEO-in used
co ns a ch-based ilm in his s udy a he lowe
spec um o he ange o alues. This means
ha he addi ion o LEO does no signi ican ly
in luence he elonga ion a b eak. The elonga ion
a b eak migh be in luenced mo e ema kably by
changes in he amoun o plas icize (i.e., glyce ol)
ela i e o co ns a ch which un o una ely was
no wi hin he scope o his s udy.
Su ace colo p ope ies
Table 3 shows he mean o he colo
pa ame e s L* (0 o 50 is conside ed da k while
51 o 100 is conside ed ligh ), a* (posi i e alues
indica e edness while nega i e alues indica e
g eenness), b* (posi i e alues indica e yellowness
while nega i e alues indica e blueness),
and WI o ilms p epa ed wi h di e en LEO
ea men s. The e we e no signi ican di e ences
in b igh ness (L*) and ligh ansmi ance in
e ms o WI alues o he ilms. The con ol (0%
LEO) ilms we e b igh e and mo e anspa en
han all he ea ed ilms. A he lowes LEO
ea men (0.5% LEO), a signi ican di e ence in
he colo o he ilms wi h ega d o hei g eenish
hue was obse ed. A 1% LEO, he yellowish
hue was signi ican ly di e en om he con ol.
These changes in colo p ope y o LEO- ea ed
ilms may be a ibu ed o ligh sca e ing caused
by lipid d ople s in he ilm ne wo k (Wang e
al., 2020). All in all, compa ed wi h he con ol,
and despi e he di e ences in colo , he LEO-
ea ed ilms mee consume equi emen s o
ood packaging ma e ials. Colo is a key ac o
conside ed in ood packaging ilms, as his can
in luence consume pe cep ion and accep ance o
ood p oduc s.
Mois u e Con en
The inco po a ion o LEO showed a signi ican
inc ease in he mois u e con en o LEO- ea ed
ilms (Table 3). Simila esul s in EO- ea ed
ilms we e epo ed by Wang e al. (2020), U ami
e al. (2019), Hossenei e al. (2009), Resianing um
e al. (2016), and do E angelo e al. (2019). The
inc ease in mois u e con en o ilms ea ed wi h
EO can be a ibu ed o he o ma ion o po ous
s uc u es ha acili a e he en y o wa e
molecules be ween polyme chains (do E angelo
e al., 2019). This allows wa e molecules o
accumula e and deposi be ween polyme chains
ia hyd ogen bonding. The wide di e ence in
molecula weigh o plas icize and s a ch can
also cause an inc ease in mois u e con en , as
g ea e gaps be ween molecula weigh s make
o bigge space be ween molecules ha can
be occupied by wa e molecules. The mois u e
con en o ilms is an impo an pa ame e ha
mus be measu ed and educed o a minimum, as
his in luences he g ow h o mic oo ganisms and
he shel li e o ood p oduc s.
An ibac e ial ac i i y o ilms
In his s udy, he disk di usion assay was
p epa ed o e alua e he an imic obial ac i i y
o co ns a ch-based ilms inco po a ed wi h LEO
agains S. au eus and S. yphimu ium (Figu e
2). The mic obial inhibi ion e ec o he ilms
is indica ed by he clea zone o med a ound
he ilms, wi h a wide clea zone sugges ing a
s onge an imic obial ac i i y. Da is and S ou
(1971), as ci ed by Resianing um e al. (2016),
ca ego ized he inhibi ion powe c i e ia in e ms
o he diame e o inhibi ion zone as ollows: e y
s ong inhibi ion (≥ 20 mm), s ong (10-20 mm),
medium (5-10 mm), and weak (≤ 5 mm). Based
on hese c i e ia, he con ol and all LEO- ea ed
ilms exhibi e y s ong an ibac e ial ac i i y
agains bo h S. au eus and S. yphimu ium, whe e
e e y inhibi ion zone is mo e han 20 mm (Table
4). One impo an inding is ha he con ol ilm
possesses s ong inhe en an imic obial p ope y
as indica ed by sizable inhibi ion zones (32.78 ±
4.60 mm o S. au eus; 29.33 ± 1.94 mm o S.
yphimu ium). This ou come may be a ibu ed o
he glyce ol and inega in he ilm o mula ion.
Ne e heless, he inhibi ion zones o he con ol
and LEO- ea ed ilms do no di e s a is ically
om each o he . I may be no ed, howe e , ha
he inhibi ion zone diame e o ilms wi h 2%
LEO agains S. au eus is conside ably highe
han hose o he o he ilms.
59
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Bo h glyce ol and inega possess
an imic obial p ope ies. In Schlie e and
Pe e son’s (2012) s udy on he an ibac e ial
ac i i y o glyce ol monolau a e in b o h and
bio ilm cul u es, i was epo ed ha glyce ol
monolau a e has po en ac i i y agains G am-
posi i e bac e ia including S. au eus, and is a
bac e icidal o a wide ange o po en ial bac e ial
pa hogens. In a s udy whe ein glyce ol is a
componen o he ilm o mula ion, U ami e al.
(2019) epo ed an imic obial ac i i y in apioca-
based edible ilms when cinnamon essen ial oil
was no inco po a ed in he ilm. On he o he
hand, inega is ich in phenolic compounds
and is well known o ha e an ioxidan and
an imic obial po en ial. Baki e al. (2017) es ed
he an imic obial ac i i y o 18 inega samples
agains G am-nega i e S. yphimu ium, E. coli
and S. au eus, and epo ed ha all samples
exhibi ed an ibac e ial ac i i y agains hese
bac e ia wi h zones o inhibi ion anging om
8.58 o 15.81 mm.
The LEO- ea ed ilms ended o ha e a
sligh ly la ge zone o inhibi ion agains he
G am-posi i e bac e ia (S. Au eus) compa ed wi h
he G am-nega i e bac e ia (S. yphimu ium).
A simila ou come was obse ed by Wang e
al. (2020), whe ein he inhibi o y zones o ilms
inco po a ed wi h Zan hoxylum bungeanum
essen ial oil agains G am-posi i e bac e ia (S.
au eus and L. monocy ogenes) we e la ge han
hose agains G am-nega i e bac e ia (E. coli).
The an ibac e ial ac i i y o LEO is due o an
in e ac ion be ween he main oil cons i uen s
and he bac e ial cell memb ane. LEO con ains
lipophilic e penes ha can change he luidi y
and pe meabili y o mic obial memb ane
o change he in acellula pH and ATP
concen a ions, which esul in cell up u e ha
inhibi s mic obial g ow h (Majewska e al., 2019).
The ou e memb ane o G am-nega i e bac e ia
con ains lipopolysaccha ide molecules ha limi
he di usion o hyd ophobic compounds. In
con as , G am-posi i e bac e ia ha e a hick
pep idoglycan laye , which can ac as p e en i e
ba ie o ce ain essen ial oils (Bu , 2004, as
ci ed by Wang e al., 2020).
Limi a ions and u u e di ec ions
In his s udy, he anspa ency o ilm
was indi ec ly measu ed by de e mining he
whi eness index (WI). WI was used o measu e he
colo shi and depa u e om whi eness b ough
on by he addi ion o he yellowish LEO. Mo e
accu a e measu emen s o anspa ency can be
done by measu ing luminous ansmi ance
and haze, ypically wi h ASTM D1003. O he
han he de e mina ion o ilm anspa ency,
he ollowing ac i i ies a e sugges ed o u u e
s udy: measu emen o ba ie p ope ies (i.e.,
wa e apo and oxygen ansmission a e);
mo phological cha ac e iza ion ia Scanning
Elec on Mic oscopy (SEM) o isualize oil
d ople dispe sion; quan i a i e an imic obial
assessmen using b o h mic odilu ion (MIC)
assays; senso y e alua ion o he ilm’s impac on
packaged ood p oduc s; and, assessmen o he
ilm’s biodeg adabili y.
Conclusion
Lemong ass essen ial oil (LEO) was
success ully inco po a ed in o co ns a ch-based
ilms. The e ec o LEO on he mechanical and
physical p ope ies o he ilms depends on he
amoun inco po a ed in he ilm, e.g., ensile
s eng h and lexibili y inc eased a 0.5% LEO
bu dec eased a highe LEO ea men s. The
base ilm exhibi s po en inhe en an imic obial
ac i i y due o glyce ol and inega in i s
o mula ion. This al eady s ong ac i i y was
only sligh ly enhanced by he addi ion o LEO.
This ou come was p obably in luenced by he
disk di usion assay’s limi a ions o hyd ophobic
compounds.
Re e ences
ASTM D882 (S anda d Tes Me hod o Tensile
P ope ies o Thin Plas ic Shee ing). (2010).
ASTM In e na ional. Wes Conshohocken,
PA.
ASTM D618 (S anda d P ac ice o Condi ioning
Plas ics o Tes ing). (2021). ASTM
In e na ional. Wes Conshohocken, PA.
A a es, L. & Chi al , A. (2016). Essen ial oils as
