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8/13/2019 Development of Antimicrobial Cellulose Packaging Through
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Available online at www.sciencedirect.com
Enzyme and Microbial Technology 43 (2008) 84–92
Development of antimicrobial cellulose packaging throughlaccase-mediated grafting of phenolic compounds
G. Elegir a,∗, A. Kindl a, P. Sadocco a, M. Orlandi b
a Stazione Sperimentale Carta Cartoni e Paste per Carta, Piazza L. Da Vinci 16, 20133 Milano, Italyb Dipartimento di Scienze dell’Ambiente e del Territorio, Universita di Milano-Bicocca, Piazza della Scienza 1, I-20126 Milano, Italy
Received 27 June 2007; received in revised form 19 September 2007; accepted 3 October 2007
Abstract
Laccase polymerization of caffeic acid and isoeugenol was shown to enhance their antimicrobial activity versus Staphylococcus aureus and Escherichia coli in liquid media. Unbleached kraft liner fibres were reacted with laccase in the presence of different phenol compounds possessing
antimicrobial activity to increase their efficacy through a covalent binding with the lignin present on the fibres. The handsheet paper obtained by
laccase antibacterial surface process (LASP) showed a greater efficacy against Gram positive and Gram negative bacteria than handsheet paper
treated only with monomeric phenol derivatives. Antimicrobial activity was function of grafted structure, time of the treatment and concentration
of phenol derivatives. In this paper several phenol compounds were tested: acids, essential oils components and dopamine. LASP in the presence
of caffeic acid or p-hydroxybenzoic acid produced paper handsheets with strong bactericidal effect on S. aureus even at low phenol monomer
concentration (4 mM), whereas a higher concentration of the monomer in the reaction mixture was required to kill E. coli. Among the tested
essential oils compounds, isoeugenol was the most effective: isoeugenol/LASP, besides killing S. aureus, showed a bacteriostatic effect on the more
resistant spore forming Bacillus subtilis. LASP in the presence of dopamine was effective against Gram positive and Gram negative bacteria. The
grafting of laccase polymerized oligomeric phenolic structures onto the fibre surface might be partially responsible of the enhanced antibacterial
activity displayed by LASP handsheet paper versus the paper treated only with monomeric phenols.
© 2007 Elsevier Inc. All rights reserved.
Keywords: Laccase; Antibacterial paper; Antimicrobial activity; Phenols; Essential oils
1. Introduction
The development of food packaging materials is mainly
intended to specifically prevent the deterioration of food,
prolonging the shelf life of packed goods and guarantee-
ing consumer safety. In recent years antimicrobial packaging
has attracted much attention from the food industry due to
the increase in consumer demand for minimally processed,
preservative-free products. Current trends suggest that inno-
vative food packaging will address active solutions where the
preservative agents will be directly applied not to the food but
to the packaging, thus only a minimal quantity of preservative
will come into contact with food [1].
Abbreviations: LASP, laccase antibacterial surface process; HBA, p-
hydroxybenzoic acid; CA, caffeic acid; GA, gallic acid; DOPA, dopamine.∗ Corresponding author. Tel.: +39 02 23955327; fax: +39 02 2365039.
E-mail address: [email protected] (G. Elegir).
Antibacterial activity of lignocellulosic fibre based products
may represent a main functional property not only for advanced
food packaging but also for hygiene paper applications. Ligno-
cellulosic fibres usually display a very low microbial resistance
and, in the case of secondary fibre based products, microbial
contaminations might be an additional issue to be taken into
account.
The increasing demand for an efficient microbial contam-
ination control in different sectors has boost a wide use of
antibiotics and biocides that has resulted in the selection of
resistant microorganisms and the build up of antimicrobial agent
residues in the environment. As a consequence, the recent years
have also witnessed a revival of the interest for natural bioactive
preservatives capable of controlling microbial contamination in
medicine, food and cosmetic applications due to their fewer
side effects and lower toxicity [2]. Thus they hold a great
potential and represent a valuable alternative and new chal-
lenges for the future to keep under control microbial contamina-
tion.
0141-0229/$ – see front matter © 2007 Elsevier Inc. All rights reserved.
doi:10.1016/j.enzmictec.2007.10.003
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G. Elegir et al. / Enzyme and Microbial Technology 43 (2008) 84–92 85
Fibre modification by an eco-friendly approach, such as the
enzymatic grafting of natural antimicrobial organic molecules to
lignocellulosic fibres, can represent a valid solution to meet the
growing consumers’ expectation respect higher hygiene stan-
dards and safer products together with environment protection
concerns.
Laccase (EC 1.10.3.2), a blue copper oxidase capable of
reacting with a large variety of aromatic substrates [3], repre-
sents a powerful tool for lignocellulosic fibre modification. In
the last decade several authors have shown that laccase treat-
ments can improve physical properties of different fibres by
producing phenoxy radicals in the lignin matrix that undergo
cross-linking reactions [4–6]. The significant amount of sur-
face lignin [7] present in high yield kraft pulp also allows the
grafting of aromatic compounds onto the fibres thus enhancing
fibre properties [8] and/or imparting completely new proper-
ties to the fibres [9]. p-Hydroxybenzoic acid (HBA) and gallic
acid (GA) have been successfully grafted onto the fibre surface
significantly increasing the number of carboxylic acid groups
[8,10].Several phenolic compounds extracted from natural sources
have been shown to exert antimicrobial activity against a wide
spectrum of microorganisms [11–13]; the antibacterial activity
has been associated with phenolic acids present in these extracts
[14,15]. Essential oils also represent a very well-known class
of natural compounds that contains different phenolic structures
particularly active on bacteria [16], even on various antibiotic
resistant ones [17]. Their mechanism of action is not yet fully
elucidated being highly dependent on the type of microorganism
and the specific chemical structures of the oil components. The
highest activityis usually reported for phenolic componentssuch
as eugenol, thymoland carvacrol andit hasbeen associated to the
acidic nature of their hydroxyl group [18,19]. Thymol and car-
vacrol were demonstrated to be active against bacteria in upper
respiratory tracts infections [20], and eugenol is widely used
in the dental field (i.e. toothpastes). Several of these phenolic
structures can react to different extent with laccase and there-
fore potentially be grafted on the lignocellulosic fibre surface to
develop covalently bound antimicrobial fibre based products.
In this work bioactive phenolic compounds were grafted
onto the surface of unbleached kraft liner fibres using a laccase
from Trametes pubescens with the aim of obtaining predictable
antimicrobial active fibre surfaces against a wide variety of
microbes.
2. Materials and methods
2.1. Chemicals
All microbiology reagents such as amino acids, peptones, extracts and
agarized media came from Oxoid, except for peptone from meat (Fluka), d-
glucose (Fluka) and l-histidine (Merck).
Fig. 1. Structure of the phenolic compounds used for the LASP in this paper.
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Salts and other chemicals came from Merck, except for NaCl (Backer);
sodium thiosulfate (Carlo Erba); l--phosphatydilcoline (Sigma–Aldrich).
The phenol compounds used for grafting are illustrated in Fig. 1. They were
all purchased from Sigma–Aldrich, except for thymol (Rectapur) and dopamine
(Fluka). They were all used as received.
2.2. Stock cultures and culture media
T. pubescens CBS 696.94 was purchased at CBS (Centraalalbureau voor
Schimmelcultures , Netherland). All other microbial strains cited in the paper
were provided by DSMZ, Deutsche Sammlung von Mikroorganismen und Zel-
lkulturen GmbH (German Collection of Microorganisms and Cell Cultures).
Bacillus subtilis ATCC 6633 (DSM 347), Escherichia coli ATCC 10536,
Enterococcus hirae ATCC 8043, Klebsiella pneumoniae ATCC 4352 (DSM
789), Staphylococcus aureus ATCC 6538 (DSM 799) and Staphylococcus epi-
dermidis ATCC 12228 (DSM 1798) were maintained frozen (−80 ◦C) and
transferred monthly on TSA (Tryptone Soya Agar) made of 15 g/l tryptone;
5 g/l soya peptone; 5 g/l NaCl and 15g/l neutralised bacteriological agar.
2.3. Production of laccase and determination of the activity
The laccase used was produced by fermentation of T. pubescens (CBS
696.94) according to Galhaup et al. [21], using CuSO4 × 5H2O as laccase pro-
duction inducer added after 48 h growth. The preinoculum was prepared from a
T. pubescens batch culture grown in a medium containing 20 g/l glucose, 10g/l
peptone from meat and 1 g/l MgSO4. After 15 days fermentation from Cu2+
induction (maximum laccase production) at 30 ◦C the culture was centrifuged
(9000× g, 30 min, 4◦C) and the supernatant containing the laccase activity was
frozen at −20 ◦C and thawed twice to precipitate the polysaccharides contained
in the media. The polysaccharide fraction was eliminated by centrifugation at
9000× g, 20 min, 4 ◦C. The supernatant was concentrated by ultrafiltration on
Amicon PM10 membrane with a molecular cut-off of 10 kDa (Danvers, MA,
USA) andexchangedin 20mM sodiumacetatebufferpH 5.Laccasewas purified
by anion exchange chromatography on a Protein-Pack Q 8HR column (Waters)
using an HPLC apparatus(Waters 600) equipped with a photodiode array detec-
tor. The column was equilibrated in 20 mM sodium acetate buffer pH 5 at a flow
rate of 1 ml/min. The proteins separation was performed running the samples
for 30 min under isocratic conditions followed by a linear NaCl gradient up to
0.25 M in 60 min. The elution of protein and laccase was monitored by 280 and
610 nm profiles, respectively. Two main laccase bound fractions (L1 and L2),
constituting approximately 65% of total laccase activity, were pooled and used
in our work.
The activity of the laccase was determined by monitoring the oxidation
of 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) at 420 nm
(ε =3.6× 104 cm−1 mol−1 l) asreportedby Fukushimaand Kirk [22]. The assay
reaction mixture contained 1mmol l−1 ABTS in 50mmol l−1 citrate–phosphate
buffer at pH 5 and a suitable amount of enzyme. Enzyme activity was expressed
in units, U, defined as mol of ABTS oxidized per min.
2.4. Oligomers preparation by laccase treatment
Isoeugenol, p-hydroxybenzoic acid and caffeic acid oligomers were dis-solved at 1 mM concentration in 250ml citrate–phosphate buffer 0.2 M pH
5, then reacted with 75 U of laccase for 4 h at 50 ◦C under constant stirring.
The reaction mixtures were ultrafiltrated on 1 kDa molecular cut-off mem-
brane (Amicon YM2, Danvers, MA, USA), using three volumes of 50 mM
citrate–phosphate buffer pH 5 to eliminate the unreacted monomers. The flux
during theultrafiltrationwas approximately0.3 ml/min. Theretentatecontaining
the oligomers was first frozen and then lyophilized at −80 ◦C. The oligomers
were maintained at −20 ◦C until use.
2.5. Determination of phenol oligomers antimicrobial activity in
liquid media
The laccase polymerized phenol oligomers were thawed and suspended in
25 ml of nutrient broth (NB) [1 g/l beef extract; 2 g/l yeast extract; 5 g/l neu-
tralised bacteriological peptone and 5 g/l NaCl] at 1 mM final concentration.
The corresponding phenol monomers were dissolved accordingly at 1 mM con-
centration in the same media. An overnight (16–18 h) bacteria pre-inoculum
culture was used toinoculate 125ml flasks containing 25ml of NB,keptat 37◦C
and 100 rpm for 24 h. The initial bacterial concentration was approximately
105 CFU/ml. Liquid bacterial cultures containing 1 mM monomers/oligomers
were compared to reference cultures without chemical additives. Once every
2 h, 1 ml of the bacterial culture was withdrawn under sterile conditions, diluted
in isotonic solution and plated in duplicate by inclusion in plate count agar(PCA, containing tryptone 5 g/l; yeast extract 2.5 g/l; glucose 1 g/l and agar
9 g/l) medium. Plates were incubated 24 h at the optimal bacterial growth tem-
perature before counting the bacterial colonies. The antibacterial effect of the
different additives was evaluated by the changes of bacterial duplication time
(t d) in the presence or in the absence of antimicrobials.
Bacterial t d was calculated from the growth constant (k ) as t d =ln2/ k . The k
constant was obtained as follows:
k =ln N 2 − ln N 1
t 2 − t 1
where N 2 is the cell number at t 2 time and N 1 is the cell number at t 1 time.
2.6. Pulp and handsheet paper preparation
A softwood kraft pulp (made out of a mixture of Pinus sylvestris and Picea
abies chips, with a kappa number of 86 and a Klason lignin content of 12.1%)
was kindly provided by the kraft liner mill Kappa Kraftliner Pitea, Sweden. The
pulp was never-dried and carefully washed with de-ionised water prior to use.
Kappa number and Klason lignin were measured according to TAPPI Methods
T 236 and T 222, respectively.
Onehundred gramspulpwerehomogenized in2 l water usinga pulper AG04
(Estanit GmbH, Germany). Handsheets (grammage: 140 g/m2) were prepared
using a conventional sheet-former, according to EN ISO 5269-1-2005 but the
pressing was performed at 6 bar (instead of 410kPa). Handsheets were dried 1 h
at 90 ◦C then conditioned at 23 ◦C and 50% humidity to constant weight for one
night before grafting reactions.
2.7. Laccase antibacterial surface process (LASP): grafting of
antibacterial chemicals on handsheet paper surface
The grafting of phenol compounds was performed by dip coating keeping
the handsheets (diameter 16 cm, approximately 2.8 g) overnight for 18 h (if not
differently specified), in a glass basincontaining the monomeric compounds dis-
solved in 75 ml 0.2 M citrate–phosphate buffer at pH 5 under constant shaking
(100rpm) at 50 ◦C. Laccase was added at 15 U/g of paper whereas the con-
centration of phenol compounds was function of their solubility: caffeic, gallic,
p-hydroxybenzoic acids anddopamine wereused up to 60 mM whereas essential
oil components (eugenol, isoeugenol and thymol) up to 4 mM. Control samples
were treated in the same way without adding the enzyme. Afterwards, the hand-
sheets were washed three times with 100 ml of distilled water for 10min, then
dried and partially sterilised at 90 ◦C for 1h.
2.8. Determination of antibacterial activity of handsheet treated
samples
A modified procedure of AATCC Test Method 100-1998 was used to
assess the antibacterial activity of handsheet treated samples. All bacterial
pre-inoculum cultures were grown overnight at 37 ◦C in 20 ml NB (horizontal
shaking at100 rpm) with theexceptionof B. subtilis that was grownat 30 ◦C.The
bacteria pre-inocula were diluted with NB medium (NB 25% or NB 12.5%) and
200l aliquots (containing 103 CFU) were used to inoculate handsheet spec-
imens (2.5 cm× 2.5cm) by the deposition of several micro-droplets on their
surface. The paper specimens were previously laid down in 60 mm petri dishes
that were placed, without cover, into 90 mm petri dishes containing approxi-
mately 15 ml of sterile water to avoid the drying of the paper specimens during
incubation. Thegrowth medium (NBused to diluteand suspend inoculum cells)
was, respectively, NB 25% for Gram positive bacteria (1 NB volume:3 sterile
isotonic solution volumes) and NB 12.5% for Gram negative bacteria (1 NB
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G. Elegir et al. / Enzyme and Microbial Technology 43 (2008) 84–92 87
volume:7 sterile isotonic solution volumes) (isotonic solution: NaCl 8.5%). For
all samples, untreated control and treated samples, three paper specimens were
inoculated with each bacteria: one specimen was used to verify the number of
inoculated bacteria (CFU T0), andthe other two to determinethe numberof cells
at the end of the antibacterial test (CFU T 24). Immediately after inoculation the
first specimen was extracted with 50 ml of neutralising solution to recover inoc-
ulated bacteria (CFU T0 determination), while the petri dishes containing the
other two inoculated specimens were incubated overnight at 37 ◦C for all bac-
teria with the exception of B. subtilis (30 ◦C). The neutralising solution had thefollowing composition: 3 g/l l--phosphatydilcoline; 5 g/l sodium thiosulfate;
1g/l l-histidine; 30g/l Tween 80; 10ml/l pH 7 buffer (34 g/l KH2PO4), the final
pH of the neutralising solution was adjusted at 7.2± 0.2 before sterilization.
After 24 h incubation the test specimens were extracted with the neutralising
solution and CFU T24 was determined. CFU values of the extraction neutral-
ising solutions were determined by serial dilution plated by inclusion in PCA
medium.
To evaluate the antibacterial efficacy of the treated samples, the CFU T 24
values were used to calculate the bacterial log reduction values by the following
formula:
log reduction = log CFU T24 untreated sample − log CFU T24 treated sample.
Due to the intrinsic variability of the antibacterial test results, at least a
2 log reduction was considered necessary to claim an antibacterial activity, as
reported in the JIS Z 2801:2000. Two different antibacterial effects could be
distinguished:
• bacteriostatic: inhibition of bacterial growth, at least 2 log reduction respect
untreated sample at T24 (CFU T24 untreated sample);
• bactericidal: inhibition of bacterial growth and concomitant reduction of the
number of inoculated bacteria (at least 2 log reduction respect the inoculated
bacteria, CFU T0). Since the initial bacteria load was approximately equal to
103 CFUfor alltests, to claim a bactericidaleffect thetreated samples should
reach a log CFU T24 value of 1.
2.9. Titration of acid groups
Conductimetric titrations were performed as described by Katz et al. [23]
to assess the amount of carboxyl groups grafted onto the fibres. Five grams of untreated and laccase-treated handsheets were disintegrated, soaked twice in
HCl 0.1 M for 45 min and washed with Milli-Q water to constant conduction
values. Then, fibres were drained and dispersed in 450ml of 0.001M NaCl.
Titration was carried out with NaOH 0.1M, while the suspension was stirred
under nitrogen atmosphere. The alkali solution was added at a rate of 0.5 ml
every 5 min, in order to allow sufficient time for equilibrium.
2.10. Size exclusion chromatography
Size exclusion chromatography (SEC) was used to evaluate the molecular
size of oligomers. The analyses were performed using Waters 600 E liquid
chromatograph connected with an HP 1040 ultraviolet diode array with a UV
detector setat a wavelength of 280 nm. TheGP-column wasan Agilent PL 3m
MIXED gel E MW 220–400 W. The acetylated lignin samples were dissolved
in tetrahydrofuran (THF), this solvent was also used as a mobile phase and the
flow rate was0.8ml min−1. Linear polystyrenestandards withmolecular weights
between 162 and 115,000g mol−1 were used to estimate the molecular weight
of the samples. The polystyrene–calibration curve was tested using acetylated
dimeric, tetrameric, and hexameric lignin model compounds.
The analysis and the evaluation of number-average molecular weight (Mn)
and weight-average molecular weight (Mw) were performed following the
methodology developed by Himmel et al. [24].
2.11. 31P NMR analysis
Resonance analysis of phosphorus (31P NMR) of derivatised oligomers
was performed in order to characterise and quantify all the different functional
groups with labile OH. In order to perform phosphorus analysis, the oligomers
were derivatised with 2-chloro-4,4,5,5-tetramethyl-1,3,2-dioxaphospholane as
reportedin literatureby Granata andArgyropoulos [25] andSaakeetal. [26]. The31P spectra were recordedusinga VarianMercury400 MHz instrumentat 333 K.
Accurately weighted phosphorilated oligomer samples (30 mg) were dissolved
in a solvent mixture composed by pyridine and deuterated chloroform 1.6:1, v/v
ratio (0.5 ml). The phospholane (100l) was then added, together with an inter-
nal standard and the relaxation reagent solution (100 l each). The 31 P NMR
data reported in this article are averages of three phosphitylation experiments.
The maximum standard deviation of the reported data was 2 ×10−2 mmol/g,
while the maximum standard error was 1 ×10−2 mmol/g.
2.12. 13C NMR
1D 13 C spectra of acetylated isoeugenol oligomers were recorded using a
VarianMercury400 MHzinstrument at 308K. The chemicalshifts werereferred
to the solvent signal at 39.5 ppm. Relaxation delay of 10 s was used between the
scans. Line broadening of 2–5 Hz wasappliedto FIDs beforeFourier transform.
For each spectrum, typically about 8000 scans were accumulated.
2.13. FT-IR spectroscopic measurements
Infrared spectra were recorded at room temperature with a Nicolett Avatar
360 FT-IR spectrometer.
3. Results
The molecular weight of three oligomers obtained by lac-
case polymerization, determined by SEC analysis, is reported
in Table 1. According to these results a higher average molecu-
lar weight was obtained when caffeic acid (CA) and isoeugenol
were reacted with laccase in comparison with p-hydroxybenzoic
acid (HBA). Yet isoeugenol showed the greater polydisper-
sity as indicated by its Mn /Mw ratio. Oligomers from CA and
isoeugenol were used for a preliminary investigation of their
antibacterial effect in liquid media. Bacterial growth of S. aureus
and E. coli in the presence of the oligomers and the corre-sponding monomers was monitored for 24 h, their duplication
times (t d) are reported in Table 2. As can be observed from
the data the antibacterial activity of the phenol derivatives was
strongly enhanced by laccase polymerization of the substrates.
Laccase control as well as CA and isoeugenol monomers at
1 mM concentration had a limited effect on bacterial growth: in
our experimental conditions S. aureus t d increased from 0.65
to 1.0 h in the presence of isoeugenol and to 0.8 h with caffeic
acid. On the contrary, the corresponding oligomers were much
more effective at the same concentration (1 mM): the isoeugenol
oligomer enhanced the t d value up to 2.3 h whereas a com-
plete growth inhibition was detected in the presence of the CAoligomer. The same results were obtained in the presence of CA
oligomer on E. coli.
Table 1
Average molecular weight distribution of oligomeric compounds obtained by
4 h laccase polymerization of the corresponding phenolic monomers
Oligomers Caffeic acid p-hydroxybenzoic acid Isoeugenol
Mw (Da) 4934 1857 3652
Mn (Da) 2596 1101 1530
Mw /Mn 1.9 1.7 2.4
Data were obtained by size exclusion chromatography (SEC) performed in
tetrahydrofuran.
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88 G. Elegir et al. / Enzyme and Microbial Technology 43 (2008) 84–92
Table 2
Comparison of bacterial growth (t d) in the presence of phenol monomers and
oligomers obtained by laccase polymerization
Duplication time, t d (h)
Staphylococcus aureus Escherichia coli
Control cellsa 0.65 0.52
Control laccaseb
0.70 0.57Caffeic acid 0.80 0.57
Caffeic acid oligomer No growth No growth
Isoeugenol 1.00 Not determined
Isoeugenol oligomer 2.30 Not determined
Monomers and oligomers were used at the same concentration (1 mM).a No phenols were added in the bacterial cultures.b Control carried out in the presence of the same amount of laccase used to
polymerize the substrates.
Themolecular structure of isoeugenol and CA oligomer were
further investigated by FTIR and 13 C NMR/ 31P NMR studies.
The acid groups content in the CA oligomer measured by 31P
NMR was 3.5 mmol/g showing that the ratio of carboxyl groups
per aromatic unit did not change during polymerization.
Caffeic acid oligomer showed some peculiar and interesting
features. The FTIR spectrum of the caffeic oligomer reported in
Fig. 2 exhibits a different profile compared to the monomer.
The benzene ring fingerprints were different in CA and CA
oligomer, the absorption peaks between 1450 and 1600 cm−1,
associated with the aromatic ring C C stretching vibration
bands, were still present, but the vibration bands of the car-
boxylic group in the oligomer appeared at 1700 cm−1 instead
of 1660cm−1. Moreover, the absorption peak at 1274 cm−1
attributed to the C–O stretching vibration bands was not any-
more present in the spectra of the oligomer. These resultsalong with NMR data (not shown) suggest that the formation
of the oligomeric product proceeded differently than perox-
idase polymerization reported by Xu et al. [27] where only
C–C ring coupling was found. Instead the structure of our CA
oligomer showed also the presence of lignin-like ether bonds.
The structure of isoeugenol oligomer resembled that of lignin
with respect to functional groups and intermonomeric link-
ages. Phenol hydroxyl decreased during polymerization due to
phenoxy radicals coupling and –O–4 intermonomeric bonds
were predominant. A significant amount of intermonomeric
–5 bonds were also detected. Based on these preliminary
results several phenol compounds known for their antimicro-
bial activity were used with laccase to covalently bind these
monomer/oligomer structures onto the fibre surface through a
radical reaction initiated by the enzyme. First results indicated
that surface treatments carried out with laccase on handsheet
paper samples (LASP) were more efficient than pulp bulk treat-
ments to impart antibacterial activity to fibres (data not shown).
Phenol derivatives were therefore reacted with laccase in the
presence of kraftliner handsheets under different conditions of
dip coating using S. aureus (Gram positive) and E. coli (Gram
negative) as main test organisms to assess antibacterial activityof paper samples.
3.1. Antibacterial fibres based on laccase grafting of
aromatic acids
Antibacterial activity of LASP treated handsheets was ini-
tially tested on S. aureus (Fig. 3). Untreated handsheets
supported a significant bacterial proliferation after 24 h con-
tact time corresponding to a S. aureus growth value (log CFU
T24 − log CFU T0) of 3.4. Control tests carried out on handsheet
paper treated only with phenol compounds in the absence of lac-
case demonstrated a slight bacteriostatic activity with HBA. Incontrast, HBA/LASP and CA/LASP samples, obtained at 4 mM
phenols concentration, showed a clear bactericidal effect on S.
Fig. 2. FTIR spectrum of (a) oligomeric caffeic acid vs. (b) caffeic acid. The laccase polymerization reaction was conducted for 4 h in the presence of 400U of
enzyme/g of caffeic acid.
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G. Elegir et al. / Enzyme and Microbial Technology 43 (2008) 84–92 89
Fig.3. Antibacterialactivity of different LASPtreatedpaperson Staphylococcus
aureus. Bacteria contact time with paper was 24 h and initial bacterial load was
logCFU T0 = 3. Bacterial growth is expressed as the logarithm of CFU T24,
the number of CFU extracted after 24 h incubation at 37◦C on paper samples.
Values shown are the mean of duplicate experiments. Antibacterial handsheets
were prepared by dip coating reacting 15 U/g laccase in the presence of various
phenolic acids at 4 mM. Symbols: () untreated control; ( ) controls treated
onlywithphenolic acids; ( ) LASPtreatedsamples. Abbreviations: CA,caffeicacid; HBA, p-hydroxybenzoic acid; GA, gallic acid.
aureus, causing the complete killing of the initially inoculated
bacterial cells whereas GA/LASP was not effective.
The same LASP treatments were tested against E. coli (data
not shown). A limited bacteriostatic effect was only detected for
the HBA/LASP samples.
The HBA/LASP antibacterial activity versus E. coli, as func-
tion of HBA concentration in the reaction media, is reported in
Fig. 4. For the paper samples prepared in the presence of lac-
case, the results clearly show that increasing the concentration
of HBA the antibacterial activity on E. coli is enhanced, reachinga significant bacteriostatic effect at 18 mM concentration, while
36 mM HBA/LASP resulted in an average bactericidal activity.
As already seen with S. aureus (Fig. 3), HBA treatment carried
Fig. 4. Effect of p-hydroxybenzoic acid (HBA) concentration on antibacterial
activity of HBA/LASP treated papers vs. Escherichia coli. Bacteria contact time
with paper was 24 h and initial bacterial load was log CFU T0 = 3. Bacterial
growth is expressed as the logarithm of CFU T24, the number of CFU extracted
after 24 h incubation at 37 ◦C on paper samples. Values shown are the mean of
duplicate experiments. Antibacterial handsheets were prepared by dip coating
reacting15 U/glaccase in thepresenceof HBA concentrations ranging from 4 to
36 mM. Symbols: () untreated control; ( ) controls treated only with HBA;
( ) LASP treated samples.
out without laccase produced a slight bacteriostatic effect on E.
coli, but only in the case of 18 mM concentration.
To obtain more knowledge regarding the effect of GA, higher
concentrations of this phenolic compound were tested in the
GA/LASP system (data not shown). The results confirmed the
absence of activity at relatively low concentrations (4–6 mM),
while at higher concentration (30 mM), 6 and 4 bacterial log
reduction values were obtained with E. coli and S. aureus,
respectively. This result shows that significant antibacterial
effects of the handsheet paper treated with GA/LASP could be
also obtained although at higher concentration than CA/LASP
and HBA/LASP.
LASP was generally performed overnight, however, since
polymerization of aromatic acids was observed after 4 h
(Table 1), the influence of reaction time on grafting and antibac-
terial effect was further investigated. The grafting efficiency of
aromatic acids was measured by titration of the acid groups
in the fibres before and after laccase reaction (15 U/g). In
the presence of CA and HBA the acid group content was
increased from 84.2mol/g (untreated kraft fibres) up to 120.6and 136.0mol/g, respectively. Increasing the time up to 24 h
or the laccase concentration up to 60 U/g did not result in any
significant improvement of the grafting.
In Fig. 5 the antibacterial activity of CA/LASP treated hand-
sheets on S. aureus versus the grafting time of CA at 4 mM
is reported. The CA handsheet samples treated for 1 h in the
presence of laccase already showed a significant bacteriostatic
activity, while after 4 and 18 h of grafting reaction they produced
a complete killing effect (bactericidal). These data suggest that
a shorter reaction time could be employed to attain antibacterial
fibres with this compound.
3.2. Antibacterial fibres based on laccase grafting of
essential oil components
Three phenolic essential oil components (eugenol,
isoeugenol and thymol) were chosen to assess their behaviour
in the LASP paper treatment. Due to their low solubility the
Fig. 5. Effect of caffeic acid (CA) grafting time on antibacterial activity of
CA/LASP treated papers vs. S. aureus. Bacteria contact time with paper was
24 h and initial bacterial load was log CFU T0 = 3. Bacterial growth is expressed
as the logarithm of CFU T24, the number of CFU extracted after 24 h incubation
at37 ◦C on paper samples. Values shown are themean of duplicate experiments.
Antibacterial handsheets were prepared by dip coating reacting 15 U/g laccase
in the presence of CA at 4 mM. Symbols: () untreated control; ( ) controls
treated only with CA; ( ) LASP treated samples.
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90 G. Elegir et al. / Enzyme and Microbial Technology 43 (2008) 84–92
Fig. 6. Antibacterial activity of different essential oils/LASP treated papers on
S. aureus. Bacteria contact time with paper was 24 h and initial bacterial load
was logCFU T0 = 3. Bacterial growth is expressed as the logarithm of CFU T24,
the number of CFU extracted after 24h incubation at 37 ◦C on paper samples.
Values shown are the mean of duplicate experiments. Antibacterial handsheets
were prepared by dip coating reacting 15 U/g laccase in the presence of vari-
ous essential oils components at 4 mM. Symbols: () untreated control; ( )
controls treated only with essential oils; ( ) LASP treated samples.
highest concentration used was 4 mM. Under these conditions
all of them were initially soluble in the buffer solution. During
the laccase catalyzed reaction, the formation of a precipitate in
the medium was observed for all the essential oil components
tested. Most likely the precipitate was due to the lower solubility
of the oligomers that were formed by laccase polymerization.
As mentioned in Table 1, isoeugenol polymerized up to 3652 Da
after 4 h reaction with laccase.
The antibacterial activity of paper grafted with essential oils
at 4 mM concentration was tested on S. aureus. Isoeugenol and
eugenol were more effective in the presence of laccase (Fig. 6)
producing a significant bacteriostatic effect, whereas thymol
demonstrated a bacteriostatic effect only in the absence of lac-case. The antibacterial efficacy of LASP in the presence of
isoeugenol at lower concentration (0.4 mM) was tested against
several Gram positive and Gram negative bacteria. Fig. 7 shows
that isoeugenol/LASP produced significant antibacterial activ-
ity only against Gram positive bacteria with the exception of
Fig. 7. Antibacterial activity of isoeugenol/LASP treated papers on different
bacteria. Bacteria contact time with paper was24 h andinitial bacterial load was
log CFU T0 = 3. Bacterial growth is expressed as the logarithm of CFU T24, the
number of CFU extracted after 24 h incubation on paper samples. Values shown
are the mean of duplicate experiments. Antibacterial handsheets were prepared
by dip coating reacting 15U/g laccase in the presence of isoeugenol at 0.4 mM.
Symbols: (
) untreated control; ( ) LASP treated samples.
Fig. 8. Antibacterial activity of DOPA/LASP treated papers on different bac-
teria. Bacteria contact time with paper was 24 h and initial bacterial load was
logCFU T0 = 3. Bacterial growth is expressed as the logarithm of CFU T24, the
number of CFU extracted after 24 h incubation on paper samples. Values shown
are the mean of duplicate experiments. Antibacterial handsheets were prepared
by dipcoatingreacting15 U/glaccase in thepresenceof DOPA at 60mM. Sym-
bols: () untreated control; ( ) controls treated only with DOPA; ( ) LASP
treated samples. Abbreviations: DOPA, dopamine.
E. hirae. In contrast to the previous test performed at 4 mM
concentration, isoeugenol/LASP handsheet paper revealed a
strong bactericidal effect on S. aureus. Bacteriostatic activity
was detected on S. epidermidis and on the more resistant spore
forming B. subtilis.
3.3. Antibacterial fibres based on laccase grafting of an
aromatic amine
A preliminary investigation on the use of aromatic amines in
theLASP is reported in Fig.8. In thiscase 60 mMdopaminecon-
centration was used during an overnight reaction with laccase.
As can be observed, dopamine treated handsheets exerted a sig-
nificant bacteriostatic effect against the Gram positive S. aureus
both with and without laccase, whereas LASP was needed to
attain antibacterial effect on all the other tested bacteria. In par-
ticular bactericidal activity was detected on the Gram positive
spore forming B. subtilis and on Gram negative E. coli, while on
K. pneumoniae only a significant bacteriostatic activity could be
claimed, due to the high log CFU T24 variability obtained. As
for aromatic acids, dopamine was found to polymerize in the
presence of laccase (data not shown).
3.4. Reproducibility of LASP treatments
The LASP efficacy was tested comparing nine different inde-
pendent replicates of the same LASP treatment. HBA/LASP at
4 mM concentration was choosen to perform this analysis. Six
replicates out of nine showed a similar log CFU T24 value, rang-
ing from 2.2 to 3.0 (data not shown) and corresponding to an
antibacterial effect of 3.9–3.1 log reduction. A low variability
of the data within a single treatment was also detected indicat-
ing a good homogeneity within the treatment. Two replicates
revealed an average log CFU T24 value close to 1 corresponding
to almost 5 log reduction but with higher variability, while only
one replicate showed a complete killing.
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