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ROMANIAN ACADEMY
“P.Poni” Institute of Macromolecular Chemistry,
Department of Physical Chemistry of Polymers
41A Grigore Ghica Voda Alley, Ro 700487 Iasi, Romania
Cornelia Vasile1, Elena Stoleru1, Bogdanel Silvestru Munteanu2, Traian
Zaharescu3, Emil Ioanid 1, Daniela Pamfil1
1 „P.Poni” Institute of Macromolecular Chemistry, Physical Chemistry of
Polymers Department, Iasi, Romania; 2“Al.I.Cuza” University, Iasi, Romania;
3 National Institute for R&D in Electrical Engineering, Bucharest, Romania
International Conference on Applications of Radiation Science and Technology (ICARST’ 2017) April, 23-28 2017
Surface modification and surface coating Most polymers used in packaging as undegradable (PE, PET ) and degradable (PLA), are chemically inert and difficult to be modified with bioactive agents Lignocellulosic materials (Chitcel-CC, Kraft paper) usually display a very low microbial resistance and therefore a frequent microbial contamination, therefore they should be protected to be safely used in food packaging.
Surface activation by corona, cold plasma in various atmospheres (air, oxygen,
nitrogen) or gamma irradiation in optima conditions of exposure was coupled with
self assembly and organisation of a polysaccharide (antimicrobial cationic or anionic polysaccharides and vegetable oils) and polyphenols, which have the
key influence on the biofunctionalization of the surfaces.
Multifunctional bioactive coating is a novel concept of active packaging. Irradiation of polymeric surface is a versatile way to implement specific functionalities which further can react with bioactive compounds in order to confer to materials antimicrobial, antifungal, antioxidant, external stimuli responsiveness and biological functions absolutely necessary to protect, to prolong self-life of food products and make them beneficial for health and to reduce the environment pollution with plastics waste and food waste.
Some Polysaccharides and Phenolic structures can react at different extent with plasma/gamma rays activated surfaces which contain implemented oxygen and nitrogen - containing groups and therefore they can be grafted onto the surface with some bioactive products such as
Chitosan, Vitamin E and C and Vegetable oils (Clove, Thyme, Tea Tree,
Rosemary, Rosehip Seeds Oil, Grape Seeds Oil, Argan Oil and Apricot Oil) with high therapeutic value.
Encapsulation/immobilization in nanostructures of the bioactive compounds by emulsion/solvent casting or co-axial electrospinning techniques.
Coating Techniques:
Solvent casting;
Immersion - Dip-coating;
Spreading/spraying;
Eelectrospinning
I) Corona (frequency 30 kHz, interelectrodes distance 7 mm, discharge
power 45 kJ/m2)
II) High-frequency plasma (O2, air or N2 were used as discharge gas); 40
Pa; 10, 20 and 30 minutes; interelectrodes distance 6.5 cm; 1.3 MHz;
discharge power of 100 W; 20-30 mA).
III) Gamma irradiation (137Cs source; irradiation doses were 5, 10, 15, 20, 30
kGy absorbed in air, at room temperature, at a dose rate of 0.4 kGy h-1.
-;-N
H2
Stability of the deposited layer onto activated
surface followed by wet chemical treatment
Using coupling agents (EDC+NHS and CDI)
leads to obtain stable chitosan + vitamin
E/vegetable oils layers chemically bonded
onto radiation activated surface. As a
consequence the bioactive compounds do
not migrate into food products and bulk
properties of base materials are not
changed.
Before After desorption
0 200 400 600 800 1000 1200 1400 1600
0,006
0,009
0,012
0,015
0,018
0,021
0,024
0,027
0,030
Pro
ton
ate
d a
min
o g
rou
ps
[mm
ol/
g]
Time [min]
PEcor,CHT/VE,pH 3.6
PEcor,EDC+NHS,CHT/VE,pH 3.6
PEcor,CDI,CHT/VE,pH 3.6
pH4 5 6 7 8 9 10 11 12
0,00
0,05
0,10
0,15
0,20
0,25
0,30
0,35
0,40
0,45
PEcor,CHT/VE
PEcor,EDC+NHS,CHT/VE
PEcor,CDI,CHT/VE
Ch
arg
e p
er m
ass
[m
mo
l/g
]
Vegetable Oils encapsulation into chitosan
or other polymers
Thickness of films: ~ 0,2 mm
I) Emulsion/solvent casting method Co-axial Electrospinning
Multifunctionality:
I. Antimicrobial
Inhibition of Bacillus cereus, Escherichia coli,
and Salmonella typhymurium grown over CHM,
CHM/Rosehip oil and CHM/Rosehip oil/C30B
Incorporation of rosehip oil and Cloisite 30B into
chitosan improved the antimicrobial activity of
chitosan film against E. coli.
Sample % Inhibition ATCC
Bacillus cereus
14579
% Inhibition
Escherichia
Coli ATCC
25922
% Inhibition
Salmonella
typhymurium 14028
24h 48h 24h 48h 24h 48h
Commercial foil PE 0 27 0 29 0 39
Chitosan MM 82 100 73 96 65 100
Chitosan MM+T80 82 100 86 100 74 100
Chitosan MM/Rosehip
oil/Rosehip oil/T80
59 100 86 100 61 94
Chitosan MM/Rosehip
oil/Rosehip oil/T80/Cloisite
C30B
82 100 90 100 68 100
Antimicrobial/antioxidant activity (%) of
CC modified with different compounds
Antibacterial activity of lignocellulose-based products represents a main functional property not only for advanced food packaging but also for hygiene items applications.
Sample Escherichia
coli
EC50, μg/mL
CC 32 -
CC/cp air 47 -
CC/cp air/Eu 79 0.886
CC/cp air/GO 48 1.828
CC/cp air/RO 85 1.097
CC/20kGy 60
CC/20 kGy/Eu 72
CC/20 kGy/GO 82
CC/20 kGy/RO 84
Antifungal (Aspergillus brasiliensis ATCC 16404, Penicillium corylophilum CBMF1
and Fusarium graminearum G87) activity was close to 100%
I. Antimicrobial
Sample composition Inhibition of
Salmonella
enteritidis
(%)
Inhibition of
Escherichia
coli (%)
Inhibition of
Listeria
monocytogenes
(%)
DPPH radical
scavenging
activity
(RSA)100mg
sample 30.min
DPPH radical
scavenging
activity
(RSA)100mg
sample 24h
PE 32 - 39 14-23 25 0 0
PEcor/CHT 100 100 92.6 0
PEcor/EDC+NHS/CHT 92.8 100 95.8 0
PEcor/CHT/0.5VE 98 100 90 27 79
PEcor/CHT/1.5VE 58 100
PEcor/CHT/3.0VE 83 100
PEcor/
EDC+NHS/CHT/VE
45 82 35
PEcor/CDI/CHT/VE 80 84 88
PEcor/
EDC+NHS/CHT/VC
100 100 100 9.2
PE/20kGy 99 91 87 5
PE/30kGy 100 100 100 13
PE/50kGy 50
PE/20kGy/EDC+NHS/CHT 100 84 96 15.1
PE/30kGy/EDC+NHS/CHT 24.3
PE/50kGy/EDC+NHS/CHT 25.7
PE/20kGy/EDC+NHS/TT 100 95 100 100
PE/30kGy/EDC+NHS/RO 100 100 100 92
Bioactive multifunctional polyethylene based food packaging with
antimicrobial activity against both gram positive and gram negative
bacteria and antioxidant activities have been obtained.
ControlPLA
PLA/N2/CHH
PLA/ N2/CHH+Clove
PLA/ N2/CHH+ARG
PLA/20kGy/CHH+Clove
PLA/20kGy/CHH+Arg0
500
1000
1500
2000
2500
Tota
l N
um
ber o
f G
erm
s [C
FU
/cm
2] TNG (CFU/cm
2), 24h
TNG (CFU/cm2), 48h
Synthetic polymeric substrates, plasma activated and /or gamma-irradiated,were tested
as active-food packaging to improve the shelf-life of the minced poultry meat, fresh
beef meet, fresh curd cheese and apple juice. The encapsulation of active vegetable
oils (antimicrobial, antioxidant, biological functions) into chitosan matrix leads to a
significant decrease of TNG when compared with the PE and PLA substrate plasma
pre-treated and surface modified only with chitosan. Clove and argan oils proved to be
valuable antimicrobial agents for delaying the spoilage of beef meat.
No significant difference are observed between plasma and gamma pre-treatment.
Testing the polymeric substrates as active-food packaging
to prolong the shelf-life of fresh beef meat
Control PE
PE/30kGy/CHH+Clove
PE/30kGy/CHH+ARG
0
500
1000
1500
2000
2500
Tota
l N
um
ber o
f G
erm
s [C
FU
/cm
2]
TNG (CFU/cm2), 24h
TNG (CFUcm2), 48h
ControlPLA
PLA/N2/CHH
PLA/ N2/CHH+Clove
PLA/ N2/CHH+ARG
PLA/20kGy/CHH+Clove
PLA/20kGy/CHH+ARG0
100
200
300
400
500
600
700
800
900
To
tal
Via
ble
Co
un
ts [
CF
U/c
m2]
24h
48h
Variation in time of Total Viable
Counts for crud cheese packed
in poly(lactic acid) modified with
chitosan and vegetable oils.
Comercial paper
BP UBPBP/CO
BP/ROUBP/CO
UBP/RO
0
1000
2000
8000
10000
12000
To
ta
l V
iab
le C
ou
nts
(u
fc
)/c
m2
24 h
48 h
Total Viable Counts of curd cheese in
presence of untreated and plasma activated
and vegetable oils modified kraft paper cold
plasma activated (a) fresh beef meat (b)
Testing the polymeric substrates as active-food packaging to
prolong the shelf-life of crud cheese
Films with rosehip oil and C30B exhibited a higher level of radical scavenging activity with values of ~ 7% compared with 1% registered for CHM film.
- Antioxidant activity higher for the
coated films
-Antioxidant activity higher for the
CHH/Clove than for CHH coated films
II. Antioxidant
Use of chitosan/vegetable oils shows synergistic activities.
0 10 20 30 40 50 60 70 80
2
3
4
5
6
7
Ab
so
rba
nce
(a
.u.)
Time [hours]
PET
PLA/10kGy
PLA/30kGy
PLA/20kGy/EDC+NHS/LF
PLA/N2/EDC+NHS/LF
0
2
4
6
8
10
12
pH
Variation in time of the absorbance
at 450 nm and pH (b) of the apple
juice in presence of lactoferrin-
PLA substrates in comparison with
juice on PET. Similar results were
obtained with PLA/ chitosan
/vegetable oils
II. Antioxidant
Sample RSA (%) Escherichia
coli
Inhibition (%)
Listeria
monocytogenes
Inhibition (%)
Salmonella
enteritidis
Inhibition (%)
PLA 0 52 40 55
PLA/cp N2 11 91 82 97
PLA/cp N2/EDC+NHS/CHT 11.8 100 100 100
PLA/cp air 12
PLA/cp air/EDC+NHS/CHT 100 100 100
PLA/10kGy 6
PLA/20kGy 8 97 100 100
PLA/20KGy/CHT 84 96 100
PLA/20KGy/EDC+NHS/CHT 100
PLA/30kGy 8
PLA/cp N2/LF 75 71 87
PLA/cpN2/EDC+NHS/LF 100 62 65 60
PLA/20KGy/EDC+NHS/LF 100 100 100
Radical scavenging activity (RSA) of
untreated, plasma and/or irradiated PLA
substrate further modified with different
bioactive compounds
III) Barrier Properties
Oxygen and carbon dioxide transmission rates are lower than that of commercial LDPE
Results: IV) pH - Responsiveness
Critical pH ≈ 6; The amino group in chitosan has a pKa value of ~6.5, which
leads to a protonation in acidic to neutral solution with a charge density
dependent on pH
Overall migration values for PLA/ATBC and PLA/CH samples (mg/dm 2).
Required by regulation (EU) no. 10/2011
<10 mg dm2 covers any food contact at
frozen and refrigerated conditions.
• Chitosan-PLA based composites were supplied to Phanerochaete
chrysosporium fungus culture medium.
• Biochemical investigation: The assay of superoxide dismutase (SOD)
activity; malondialdehyde (MDA) assayed using thiobarituric acid (TBA)
and catalase in fungi mycelium samples. All resulted numerical values
were expressed relatively to the amount of the protein fungus mycelium.
• Gel Permeation Chromatography – Variation of the average molecular
weight
• ATR-FTIR – structural modification
• Scanning Electron Microscopy (SEM) and
• Atomic Force Microscopy (AFM) –morphology change
• Soil burial test
Biodegradation
Biodegradable substrates as polylactic acid and cellulosic materials (cellulose/chitin blends and kraft paper) undergone to the same procedures gave very promising results, moreover these are easily recyclable and integrate into environment after use.
Superoxide dismutase Catalase enzyme (CAT)
Malondialdehyde Extracellular protein of P. chrysosporium
Polymeric substrate influence on Phanerochaete
chrysosporium characteristics
0
2
4
6
8
10
12
14
Pro
tein
co
nte
nt
(mg
/g) 7 days
14 days
0
1
2
3
4
5
6
7
8
SO
D A
cti
vit
y (
UC
/mg
pro
tein
)
7 days
14 days
4000 3500 30002000 1500 10000,0
0,1
0,2
0,3
0,4
0,5
0,6
1700 1600 15000,00
0,01
0,02
0,03
0,04
0,05
0,06
Ab
sorb
an
ce [
a.u
]
Wavenumber [cm-1]
PLA
PLA/14d
(a)
Ab
sorb
an
ce [
a.u
]
Wavenumber [cm-1]
4000 3500 30002000 1500 1000 500
0,00
0,05
0,10
0,15
0,20
0,25
0,30
0,35
3400 32000,000
0,005
0,010
0,015
0,020
0,025
0,030
Ab
sorb
an
ce [
a.u
.]
Wavenumber [cm-1]
PLA/30kGy
PLA/30kGy/14d
(b)
Ab
sorb
an
ce [
a.u
.]
Wavenumber [cm-1]
4000 3500 3000 2000 1500 1000
0,0
0,1
0,2
0,3
0,4
0,5
Ab
sorb
an
ce [
a.u
.]
Wavenumber [cm-1]
PLA/30kGy/CHH
PLA/30kGy/CHH/14d(c)
3100 3050 3000 2950 2900 2850 2800
0,01
0,02
0,03
Ab
sorb
an
ce [
a.u
.]
Wavenumber [cm-1]
4000 3500 3000 2000 1500 1000
0,0
0,1
0,2
0,3
0,4
0,5
0,6
1700 1650 1600 1550 15000,00
0,01
0,02
0,03
0,04
0,05
0,06
0,07
0,08
Ab
sorb
an
ce [
a.u
.]
Wavenumber [cm-1]
PLA/N2
PLA/N2/14d
(d)
Ab
sorb
an
ce [
a.u
.]
Wavenumber [cm-1]
(d)
II) new bands appear - at 3729 cm-1 - free –OH stretching, 3276 cm-1 - H-bonded –
OH stretching, 1659 cm-1 - amide C=O stretching, N-H bending at 1625 cm-1 and
1543 cm-1- to the fungal hyphae grown on the biodegraded sample’s surface. III)
The bands at 870 cm-1 and 755 cm-1 represent the amorphous and crystalline phases
of PLA, respectively – crystallinity increases
Biodegradation - ATR-FTIR results
After 14 days of
biodegradation of
PLA:
I) 1410 cm-1, which is
assigned to –CH3
vibration from amide
group, almost
disappear – chitosan
deacetylation
Average molecular weight change after degradation under Phanerochaete chrysosporium action of the PLA-based samples
Sample Mn
(x103)
g/mol
Mw
(x103)
g/mol
Mz
(x103)
g/mol
PDI
Mw/Mn
[]
mL/g
PLA 299.7 451.4 678.7 1.507 199.2
PLA/7d 39.21 76.08 136.9 1.940 99.12
PLA/14d 48.56 85.02 144.6 1.751 101.5
PLA/30kGy 49.73 88.34 153.5 1.776 99.86
PLA/30kGy/7d 26.68 48.33 81.63 1.811 60.26
PLA/30kGy/14d 27.84 46.70 75.17 1.678 58.45
PLA/N2 256.32 395.42 578.3 1.679 179.2
PLA/N2/CHH 242.75 282.48 585.4 2.305 199.2
PLA/N2/CHH/7d 171.92 168.01 385.6 2.33 224.6
PLA/N2/CHH/14d 46.25 79.80 134.6 1.725 96.72
PLA/30kGy/CHH 43.35 107.6 213.3 2.481 107.0
PLA/30kGy/CHH/7d 29.80 50.87 84.01 1.707 62.47
PLA/30kGy/CHH/14d 27.59 47.23 77.48 1.712 58.80
PLAPLA/N2
PLA/N2/EDC/CHHPLA/30kGy
PLA/30kGy/CHH
0
10
20
30
40
50
60
70
80
90
100
110
120
AF
M R
ou
gh
nes
s [n
m]
Initial
7 days exposure to P. Chrysosporium
14 days exposure to P. Chrysosporium
Modification of surface - the formation of
oligomers and other low-molecular biodegradation
products as a result of random chain scission.
Some of them can agglomerate at the surface
creating the observed grains. The most significant
topographical change in terms of morphology and
roughness is observed for the PLA sample gamma
irradiated and surface modified with chitosan
Biodegradation AFM results
Gamma-irradiation is much efficient in terms of bioactive functions
(especially antioxidant) conferred to packaging materials.
The coating/encapsulation can be performed by dip-coating,
emulsion/solvent casting or electrospraying/electrospinning
techniques. The last is the most efficient because very thin
surface layers assure both antimicrobial and antioxidant
characteristics of packaging.
Stable coating prevents migration in food product as it was
assessed by the release studies in simulated media. The incorporated natural additives do not negatively affect the
consumers health. Nanocomposites and nanostructures protect the food against environmental factors and may enhance stability and quality of food products.
The PLA/CHT stratified composites supported fungal growth of Phanerochaete chrysosporium. The presence of bioaccessible material, i.e., PLA and chitosan, facilitated degradation.The
gamma irradiated PLA samples show increased degradation. The similar results have been obtained by soil burial test.