Development of Antimicrobial Cellulose Packaging Through

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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.

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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.



86 G. Elegir et al. / Enzyme and Microbial Technology 43 (2008) 84–92

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




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.




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.




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.




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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