1
Cha ac e iza ion and an imic obial ac i i y o ac i e polyp opylene ilms con aining O egano
essen ial oil and Allium ex ac o be used in packaging o mea p oduc s.
Ma ía Llana-Ruiz-Cabello1, Sil ia Picha do1*, José Ma ía Be mudez3, Albe o Baños2, Juan José
A iza2, En ique Guillamón2, Susana Aucejo3, Ana M. Cameán1
1A ea o Toxicology, Facul y o Pha macy, Uni e sidad de Se illa, P o eso Ga cía González n°2,
41012 Se ille. Spain.
2DOMCA S.A. Camino de Jayena s/n, 18620 Alhendín, G anada, Spain.
3A ea o Packaging ma e ials and Sys ems, ITENE, C/ Albe Eins ein 1, 46980 Pa e na, Valencia,
Spain.
*Co esponding au ho :
Sil ia Picha do Sánchez
A ea o Toxicology, Facul y o Pha macy, Uni e sidad de Se illa, P o eso Ga cía González n°2,
41012 Se ille. Spain.
E-mail add ess: spicha [email protected]
Tel: +34 954 556762
Fax: +34 954 556422
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Abs ac
Cooked ham is mo e p one o spoilage han o he mea p oduc s due o i s p ope ies, making
he p ese a ion echniques a key s ep in i s comme cializa ion. One o he mos p omising
p ese a ion s a egies is he use o ac i e packaging. O egano essen ial oil (OEO) and
P oallium® (an Allium ex ac ) ha e p e iously shown o be use ul in polylac ic acid (PLA) ac i e
ilms o eady o ea salads. The p esen wo k aims o s udy he sui abili y o polyp opylene
(PP) ilms con aining OEO and P oallium® in he p ese a ion o cooked ham. Conce ning he
echnological ea u es o he s udied ma e ial, no ema kable change in he mechanical o
op ical p ope ies o PP ilms con aining he ac i e subs ances we e eco ded in compa ison o
he PP ilm wi hou ex ac s. Al hough ilms con aining bo h ac i e subs ances we e mo e
lexible han he con ol ilm and less s ong, highligh ing he plas iciza ion e ec o he na u al
ex ac s. Mo eo e , physical p ope ies changed when ac i e subs ances we e added o he
ilm. The inco po a ion o 4% P oallium® a ec ed in a highe ex end he anspa ency o he
ilm han in he case o 8% OEO, unde going a dec ease in he anspa ency o 40 and 45%,
espec i ely. Mo eo e , only he ilm con aining he highes amoun o OEO (8%) dec eased
signi ican ly he hickness. Bo h ac i e subs ances showed an ibac e ial p ope ies; howe e ,
P oallium® ac i e ilms seemed o be mo e e ec i e agains B ocho ix he mosphac a han PP
ilms con aining OEO, wi h all pe cen ages o P oallium® killing he bac e ial popula ion
p esen in he ham a e 60 days. In addi ion, ma e ials con aining he lowes P oallium®
con en exhibi ed he highe accep abili y by he consume s in he senso y analyses wi h a
willing pu chase o 63-100%, e en be e han he con ol package (56-89%). In ac , 2% o
P oallium® ob ained he bes esul s in he smell s udy pe o med by he panellis s.
Keywo ds: polyp opylene; o egano essen ial oil; Allium ex ac ; ac i e packaging
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Abb e ia ions: Analysis o a iance mul i ac o (ANOVA); B ocho h ix he mosphac a (B.
he mosphac a); colony o ming uni s (CFU); c ys allisa ion empe a u e (Tc); elas ic modulus
(E); elonga ion a b eak (EAB); en halpy o c ys alliza ion (ΔHc); en halpy o mel ing (ΔHm);
essen ial oils (EOs); e hylene inyl alcohol (EVOH); leas signi ican di e ence (LSD); mel ing
empe a u e (Tm); o egano essen ial oil (OEO); polylac ic acid (PLA); polyp opylene (PP);
p opyl hiosulphina e oxide (PTSO); Spanish Type Cul u e Collec ion (CECT); ensile s eng h
(TS); e ahyd o u an (THF); he mog a ime ic analysis (TGA).
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1. In oduc ion
Mea and mea p oduc s a e good media o he g ow h o mic oo ganism (Van Hau e e al.
2016). Cooked ham is mo e p one o spoilage han o he mea p oduc s due o i s low sal
con en , pH nea 6 and wa e ac i i y highe han 0.945. In addi ion, cooked ham is usually
sliced and packaged be o e being sold and consequen ly i s shel -li e could dec ease (Baños e
al. 2012). The e o e, he p ese a ion s a egies o hese oods a e o g ea impo ance.
New ends in consume demands, ega ding minimally p ocessed ood o eady- o-ea esh
ood, a e d i ing a sea ch o inno a i e ways o main ain ood quali y, eshness and sa e y
(Appendini and Ho chkiss 2002). One o hese new de elopmen s is ac i e packaging, which
was de ined in Regula ion (EC) No 450/2009. These ma e ials include an imic obial ac i e ilms
ha can be classi ied in wo ca ego ies: hose made by using polyme s inhe en ly
an imic obial; and hose ha in ol e he di ec inco po a ion o he an imic obial addi i e in o
he packaging ilms (Suppakul, e al. 2003). Ac i e ood packaging sys ems con aining
an imic obial addi i es a e polyme ic ma e ials de eloped o elease hese ac i e subs ances
in o he ood in o de o p e en mic obial g ow h and o ex end oods u shel -li e (Del
Nobile e al. 2009). Polyole ins ha e been used as ood packaging ma ices in he de elopmen
o ac i e ilms due o hei mechanical, ba ie , op ical and he mal p ope ies (Ramos e al.
2012; Gigli e al. 2014). Polyp opylene (PP) is a polyole in wi h low wa e apo ansmission,
medium gas pe meabili y, good esis ance o g eases and chemicals, good ab asion esis ance,
high empe a u e s abili y and good gloss and high cla i y. All o hese ea u es make PP
sui able o ood packaging (Robe son 2012; Ramos e al. 2012; Ramos e al. 2014; Mu iel-
Gale e al. 2013).
Rega ding ac i e subs ances, se e al s udies ha e shown ha consume s ha e ecen ly
become mo e in o med abou ood addi i es and end o choose hose wi h addi i es o
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na u al o igin a he han hei syn he ic analogues (Ca ocho e al. 2015). In his ega d,
essen ial oils (EOs) and plan ex ac s ha e been he a ge o ex ensi e esea ch because
hese subs ances ha e demons a ed biological p ope ies ha exhibi bene i s in ood (Llana-
Ruiz-Cabello e al. 2015; Maisanaba e al. 2017; Ribei o-San os e al. 2017). The e o e, new
al e na i es based on a oma ic plan s, such as EOs, ha e been s udied (Ramos e al. 2014). The
ad an ages obse ed o ac i e packaging con aining EOs compa ed wi h di ec addi ion o
hese addi i es o he ood, such as lowe amoun o compounds equi ed o ex ended e ec s
due o con olled mig a ion om he ilm o he ood a e inc easing he demand o hese
ma e ials (Sanchez-Sil a e al. 2014). Besides, when he addi i e is sp ayed di ec ly o ood, he
ac i e subs ances could in e ac wi h ood cons i uen s causing hei dena u aliza ion. This
phenomenon can be a oid also by using ac i e packaging con aining EOs (Sung e al. 2013).
Among EOs, o egano essen ial oil (OEO) and i s majo compounds, ca ac ol and hymol, ha e
shown i s use ulness as ac i e subs ance in ood packaging. A p e ious expe imen al s udy
pe o med in ou labo a o y ha e p o ed he sui abili y o OEO when inco po a ed o a ma ix
o polylac ic acid (PLA) as an ioxidan agen in eady- o-ea salads (Llana-Ruiz-Cabello e al.
2016). Mu iel-Gale e al. (2013) also showed he abili y o e hylene inyl alcohol (EVOH)-
coa ed PP ilms con aining OEO o imp o e he shel li e o packaged salad. Simila ly, Ramos e
al. (2012) epo ed he an imic obial ac i i y o PP ilms con aining ca ac ol and hymol
agains bac e ial s ains p esen in oods such as Esche ichia coli o S aphylococcus au eus.
Mo eo e , ano he plan ex ac , P oallium®, is a comme cial p oduc based on Allium ex ac
ha is composed o o ganosulphu compounds. Ga lic EO and i s majo o ganosulphu
compounds ha e shown an imic obial ac i i y when hey a e included in ac i e packaging
(Sung e al. 2014; Teixei a e al. 2014). Speci ically, we ha e p e iously obse ed ha ilms o
PLA con aining P oallium® showed an imic obial ac i i y agains bac e ia, yeas and molds

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(Llana-Ruiz-Cabello e al. 2015b). Howe e , his is he i s wo k dealing he use o ac i e
packaging con aining P oallium® in he p ese a ion o ham.
The aim o he p esen wo k was o de elop new ac i e ilms based on PP con aining OEO and
P oallium® and u he cha ac e ize hem hough op ical, mechanical and he mal p ope ies.
Mo eo e , he e ec i eness o hese ilms in he p ese a ion o cooked ham was also
e alua ed.
2. Ma e ials and Me hods
2.1 Supplies and Chemicals
Polyp opylene ISPLEN® PP 050 G2M was pu chased om Repsol YPF S.A. (Mad id, Spain).
O egano essen ial oil was acqui ed om El Ja pil (Alme ía, Spain). P oallium® wi h 14.5% o
PTSO (Llana-Ruiz-Cabello e al. 2015b) as ac i e agen was p o ided by Domca S.L. (Alhendin,
G anada, Spain).
Chemicals o he di e en assays we e pu chase om Sigma-Ald ich (Spain) and VWR
In e na ional Eu olab (Spain). Ham o he in i o assays was pu chase in a local ma ke
(Valencia, Spain).
2.2 Film p epa a ion
Two ypes o ac i e PP ilms we e ob ained, one o hem con aining nominal OEO con en s o
2, 5 and 8 % (w/w) and he o he con aining nominal P oallium® con en s o 2, 3 and 4 %
(w/w). Films we e p epa ed by mel blending in a win-sc ew ex ude (DSE 20-40D,
B abende , Ge many). Ex usion empe a u es we e se a 200-205 ºC wo king a a sc ew sped
o 70 min-1. Ac i e subs ances we e in oduced in he mixe once he polyme was mel ed and
we e ed in o he ba el h ough he la e al liquid po a L/D 10 o educe possible ola ili y
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and deg ada ion losses. The a e age wid h and hickness o he ilms we e a ound 10 cm and
80 μm, espec i ely.
2.3 Quan i ica ion o he o e all con en o OEO and P oallium® inco po a ed in o PP ilms
The amoun o OEO and P oallium® con ained in PP ilms was e alua ed using
The mog a ime ic Analysis (TGA). B ie ly, samples we e hea ed o 900ºC and he loss o mass
was measu ed in a he mal analyse Q5000IR (TA Ins umen s, New Cas le, DE, USA) as
desc ibed in Llana-Ruiz-Cabello e al. (2015b).
2.4 Quan i ica ion o ca ac ol, hymol and p opyl hiosulphina e oxide inco po a ed in o PP
ilms
The main cons i uen s o OEO (i.e. ca ac ol and hymol) and P oallium® (i.e. p opyl
hiosulphina e oxide, PTSO) inco po a ed in o he PP ilms we e ex ac ed using
e ahyd o u an (THF) as sol en . Then, The mo Finnigan gas ch oma og aph (GC) equipped
wi h a DB-5MS column (Agilen Technologies, USA) (30 m, 0.25 mm, ilm hickness 0.25 μm)
using helium as a ca ie gas ( low a e o 1 ml min−1) was used o he analyses o ac i e
subs ance. The quan i ica ion o ca ac ol and hymol was pe o med ollowing he me hod
desc ibed by Llana-Ruiz-Cabello e al. (2016) and he quan i ica ion o PTSO was ca ied ou
acco ding o Llana-Ruiz-Cabello e al. (2015b).
2.5 Physical p ope ies
The ligh ansmission and anspa ency o ilms we e measu eda selec ed wa eleng hs
be ween 200 and 800 nm, using a Jasco V-630 UVeVisible eco ding spec opho ome e
(Mad id, Spain) acco ding o he me hod desc ibed in Llana-Ruiz-Cabello e al. (2015b). The
anspa ency o he ilms was calcula ed acco ding o Han and Flo os (1997), as ollows:
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T anspa ency alues = (-logT600)/x; whe e T600 is he ac ional ansmi ance a 600 nm; and
x: is he ilm hickness in mm.
Thickness we e measu ed was measu ed in a leas six di e en loca ions on each ilm using a
digi al mic ome e Mi u oyo 547-400 Absolu e (Kawasaki, Japan). The esul s a e exp essed as
he median ± s anda d de ia ion.
2.6 Mechanical p ope ies
Films we e condi ioned o 48 h a 25 ºC and 50±5 % RH be o e es ing hei mechanical
p ope ies. Tensile s eng h (TS), elonga ion a b eak (EAB) and elas ic modulus (E) we e
de e mined a oom empe a u e using an M350-20CT Uni e sal Tes ing Machine
(Tes ome ic, Rochdale, England) acco ding o UNE-EN-ISO 527-1, wi h sligh modi ica ions.
2.7 The mal analysis
A Di e en ial Scanning Calo ime e DSC Q2000 (TA Ins umen s) was used o de elop he
he mal analysis o ilms. C ys allisa ion empe a u e (Tc), and mel ing empe a u e (Tm) o
he di e en PP ilms we e measu ed using a Di e en ial Scanning Calo ime e DSC Q2000 (TA
Ins umen s). Specimens weighing 6 mg we e hea ed a he a e o 10 ºC min-1 om 20 ºC o
250 ºC. Then, he samples we e cooled a 10 ºC min-1 om 250 ºC o -50 ºC. Finally, he
samples we e hea ed again a 10 ºC min-1 up o 250 ºC.
The c ys allini y was calcula ed as he a io be ween he en halpy o he mel ing poin o he
co esponding heo e ical en halpy o 100 % c ys alline PP (es ima ed as 165 J/g o PP
acco ding o Zhu (2002) in pe cen age basis.
2.8 An imic obial ac i i y in cooked ham
2.8.1 Mic obial s ain
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B ocho h ix he mosphac a, supplied by he Spanish Type Cul u e Collec ion (CECT), was g own
o e nigh on b ain hea in usion (BHI, Sha lau, Ba celona, Spain) a 28 ºC in ae obic
a mosphe e.
2.8.2 Expe imen al design
Th ee slides o ham we e placed in polys y ene ays (135 x 180 mm, BANDESUR, Alcalá la
Real, Spain) o each ea men and inocula ed wi h a suspension o B. he mophac a o yield a
inal bac e ial le el o 105 colony o ming uni s (CFU)/cm2 using a Dig alsky sp eade . In o de
o sepa a e he slides he de eloped ilms we e used as in e laye . Di e en ays we e
p epa ed using PP and PP con aining OEO (2, 5 and 8 %) o PP con aining P oallium® (2, 3 and
4 %).
Samples packaged we e s o ed in PP/polyamide plas ic bags unde acuum (Techno ip EVT-
10-2-CD-SC, Ba celona, Spain) and s o ed a 5 ºC.
The an imic obial ac i i y o PP ilms was e alua ed a e 1, 6, 12, 21, 27 and 60 days o
packaging by s udying he e olu ion o B. he mophac a popula ion in he cooked ham along
he s o age ime.
2.8.3 Mic obial analysis
Samples o he su ace o each slide o ham we e aken a selec ed imes o measu e iable
coun s o bac e ia. Each slide (ap ox. 14 g) was asep ically emo ed and homogenized wi h 125
ml o pep one wa e (0.1 % pep one) using a Mas ica o blende (IUR, Ba celona, Spain) o 1
min. Then, a decimal dilu ion se ies was p epa ed o pla ing on STAA aga (Oxoid LTD,
Basings oke, Hampshi e, England). Finally, pla es we e incuba ed ae obically a 28 ºC o 48-72
h be o e bac e ial coun s we e de e mined. Resul s we e exp essed as CFU/cm2.
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dec eased he mic obial coun s o his mic oo ganism. The e o e, he use o hese in e laye
ilms suppose a no el ool ha in combina ion wi h o he hu dles, such as e ige a ion o
acuum packaging, could ex end he shel li e o mea p oduc s.
The abili y o OEO, and i s majo compounds, inco po a ed in di e en ilm ma ices o educe
mic obial coun s in ood has been epo ed by se e al au ho s. Ramos e al. (2012) epo ed
ha PP ilms con aining 8 % o ca ac ol and hymol and ilms con aining an 8 % mix u e o
bo h subs ances showed an imic obial ac i i y agains S aphylococcus au eus and Esche ichia
coli. An imic obial ac i i y was obse ed in salad packaged in EVOH-coa ed PP ilms con aining
OEO (Mu iel-Gale e al. 2013) and polylac ic acid (PLA) ilms con aining OEO (Llana-Ruiz-
Cabello e al. 2016).
3.6 Senso y analyses
Figu e 2 shows he median sco es o each senso y desc ip o a he di e en s o age imes
and packaging ma e ial as well as he box and whiske plo o he sco es ob ained along
s o age ime o each packaging ma e ial.
Smell was in gene al he lowes sco ed desc ip o in compa ison o he o he s and hus he
mos c i ical one (Figu e 2A). The panellis s de ec ed in mos o he cases he inhe en smell o
he o egano and Allium ex ac s and hey e alua ed he smell as some hing unexpec ed. In
ac , gene al accep abili y and pu chase in en ion we e highly co ela ed o he smell.
Mul i ac o ANOVA showed a signi ican impac o bo h, packaging ma e ials and days o
s o age (p < 0.05) on he senso y pe cep ion o he smell. Ham in he con ol package was
abo e he h eshold alue o 5 om day 1 o day 11. F om ha poin , smell was sligh ly below
he h eshold up o day 60. The ac i e ma e ials con aining OEO we e signi ican ly below he
sco es gi en o he con ol package. Ma e ials con aining 3% and 4% P oallium® we e below
he con ol package al hough non-signi ican di e ences we e ound. The bes esul s o he

17
smell we e ob ained o ma e ials con aining 2% o P oallium®. This ma e ial was sco ed abo e
5 o he en i e s o age ime.
Figu e 2B shows he median sco es o isual appea ance. In his case, his senso y a ibu e
was abo e o e y close o he h eshold alue o he di e en ma e ials and s o age ime.
ANOVA iden i ied he s o age ime as non-signi ican (p > 0.05). In he case o he packaging
ma e ials, jus he ma e ial con aining 2% OEO was signi ican ly di e en om he o he
ma e ials, sco ing he wo s . Su p isingly, isual appea ance was be e e alua ed han smell,
bu sca cely in luenced in he pu chase in en ion o consume s.
Gene al accep abili y showed a simila end o he smell (Figu e 2C). ANOVA showed a
signi ican impac o he s o age ime and packaging ma e ial (p < 0.05). Accep abili y o he
ham packaged in he con ol ma e ial dec eased om day 1 o day 11 and emained close o
abo e he h eshold up o day 60. The ac i e ma e ials con aining OEO and P oallium® showed
alues in gene al below he con ol package al hough wi h e y ew signi ican di e ences. The
exemp ion was PP con aining 2% P oallium®, which sco ed he bes and abo e he h eshold
o he en i e s o age.
Finally, Table 5 shows he pe cen age o panellis s willing o pu chase o consume he ham
packaged in o he di e en ma e ials and s o age ime. The bes esul s we e ob ained o he
con ol ma e ial and he PP con aining 2% P oallium®. Pu chase in en ion was highe han 50%
o bo h ma e ials and along he en i e s o age ime. Ma e ials con aining OEO we e in gene al
below 50% including 0% pu chase in en ion a some speci ic days.
4. Conclusions
Polyp opylene (PP) ilms con aining P oallium® o OEO showed simila he mical and
mechanical p ope ies as PP. Mo eo e , P oallium® ac i e ilms seemed o be mo e e ec i e
agains B. he mosphac a han PP ilms con aining OEO. Rega ding he senso ial analyses,
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ma e ials con aining he lowes P oallium® pe cen age was highly accep ed by he consume s,
e en be e han he con ol package. Howe e , o egano la ou was no iced as s ange o
consume s, and had a nega i e impac in pu chase in en ion.
Acknowledgemen s: he au ho s wish o hank he Spanish Minis y o Science and Inno a ion
(AGL2012-38357-C02-01) co- inanced by FEDER unds and Jun a de Andalucía (AGR-7252) o
he inancial suppo o his s udy and he Mic oscopy Se ice o CITIUS. Ma ía Llana-Ruiz-
Cabello also g a e ully acknowledges Jun a de Andalucía o he p edoc o al g an associa ed
o he AGR7252 p ojec .
19
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Figu e cap ion
Figu e 1. TGA cu es ob ained o OEO, P oallium, PP ilm and PP con aining OEO and
P oallium®.
Figu e 2. Senso y analysis measu ed as smell (A), isual appea ance (B) and gene al
accep abili y (C) o cooked ham in con ol and ac i e packaging con aining OEO (2, 5, 8 %) o
P oallium® (2, 3 4 %). P o: P oallium®.
Film sample
O e all
con en (%)
Ca ac ol
con en (%)
Thymol
con en (%)
PTSO con en
(%)
PP 2% OEO
1.6 ± 0.3
1.10 ± 0.10
0.132 ± 0.011
PP 5% OEO
3.9 ± 0.2
2.9 ± 0.3
0.33 ± 0.02
PP 8% OEO
5.84 ± 0.11
4.2 ± 0.3
0.420 ± 0.011
PP 2%
P oallium
1.680 ± 0.014
0.183 ± 0.011
PP 3%
P oallium
2.56 ± 0.06
0.196 ± 0.015
PP 4%
P oallium
2.82 ± 0.10
0.25 ± 0.06
Table 1. Con en o OEO, P oallium® and hei main compounds inco po a ed in o he PP ilms.