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.

Acknowledgements: This research received financial

support from IAEA, Romanian UEFISCDI and Norway

Research Foundation