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Enzyme and Microbial Technology 43 (2008) 8492

Development of antimicrobial cellulose packaging through laccase-mediated grafting of phenolic compounds

G. Elegir a, , A. Kindl a , P. Sadocco a , M. Orlandi b
b

Stazione Sperimentale Carta Cartoni e Paste per Carta, Piazza L. Da Vinci 16, 20133 Milano, Italy Dipartimento di Scienze dellAmbiente e del Territorio, Universit` a 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 bres were reacted with laccase in the presence of different phenol compounds possessing antimicrobial activity to increase their efcacy through a covalent binding with the lignin present on the bres. The handsheet paper obtained by laccase antibacterial surface process (LASP) showed a greater efcacy 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 bre 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 specically prevent the deterioration of food, prolonging the shelf life of packed goods and guaranteeing 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 innovative 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, phydroxybenzoic acid; CA, caffeic acid; GA, gallic acid; DOPA, dopamine. Corresponding author. Tel.: +39 02 23955327; fax: +39 02 2365039. E-mail address: gelegir@sperimentalecarta.it (G. Elegir). 0141-0229/$ see front matter 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.enzmictec.2007.10.003

Antibacterial activity of lignocellulosic bre based products may represent a main functional property not only for advanced food packaging but also for hygiene paper applications. Lignocellulosic bres usually display a very low microbial resistance and, in the case of secondary bre based products, microbial contaminations might be an additional issue to be taken into account. The increasing demand for an efcient microbial contamination 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 challenges for the future to keep under control microbial contamination.

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Fibre modication by an eco-friendly approach, such as the enzymatic grafting of natural antimicrobial organic molecules to lignocellulosic bres, can represent a valid solution to meet the growing consumers expectation respect higher hygiene standards 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], represents a powerful tool for lignocellulosic bre modication. In the last decade several authors have shown that laccase treatments can improve physical properties of different bres by producing phenoxy radicals in the lignin matrix that undergo cross-linking reactions [46]. The signicant amount of surface lignin [7] present in high yield kraft pulp also allows the grafting of aromatic compounds onto the bres thus enhancing bre properties [8] and/or imparting completely new properties to the bres [9]. p-Hydroxybenzoic acid (HBA) and gallic acid (GA) have been successfully grafted onto the bre surface signicantly 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 [1113]; 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 specic chemical structures of the oil components. The highest activity is usually reported for phenolic components such as eugenol, thymol and carvacrol and it has been associated to the acidic nature of their hydroxyl group [18,19]. Thymol and carvacrol were demonstrated to be active against bacteria in upper respiratory tracts infections [20], and eugenol is widely used in the dental eld (i.e. toothpastes). Several of these phenolic structures can react to different extent with laccase and therefore potentially be grafted on the lignocellulosic bre surface to develop covalently bound antimicrobial bre based products. In this work bioactive phenolic compounds were grafted onto the surface of unbleached kraft liner bres using a laccase from Trametes pubescens with the aim of obtaining predictable antimicrobial active bre 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), dglucose (Fluka) and l-histidine (Merck).

Fig. 1. Structure of the phenolic compounds used for the LASP in this paper.

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G. Elegir et al. / Enzyme and Microbial Technology 43 (2008) 8492 tralised bacteriological peptone and 5 g/l NaCl] at 1 mM nal concentration. The corresponding phenol monomers were dissolved accordingly at 1 mM concentration in the same media. An overnight (1618 h) bacteria pre-inoculum culture was used to inoculate 125 ml asks containing 25 ml of NB, kept at 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 temperature before counting the bacterial colonies. The antibacterial effect of the different additives was evaluated by the changes of bacterial duplication time (td ) in the presence or in the absence of antimicrobials. Bacterial td was calculated from the growth constant (k) as td = ln 2/k. The k constant was obtained as follows: k= ln N2 ln N1 t2 t1

Salts and other chemicals came from Merck, except for NaCl (Backer); sodium thiosulfate (Carlo Erba); l--phosphatydilcoline (SigmaAldrich). The phenol compounds used for grafting are illustrated in Fig. 1. They were all purchased from SigmaAldrich, 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 Zellkulturen 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 epidermidis 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 15 g/l neutralised bacteriological agar.

where N2 is the cell number at t2 time and N1 is the cell number at t1 time.

2.3. Production of laccase and determination of the activity 2.6. Pulp and handsheet paper preparation
The laccase used was produced by fermentation of T. pubescens (CBS 696.94) according to Galhaup et al. [21], using CuSO4 5H2 O as laccase production 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, 10 g/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 ultraltration on Amicon PM10 membrane with a molecular cut-off of 10 kDa (Danvers, MA, USA) and exchanged in 20 mM sodium acetate buffer pH 5. Laccase was puried by anion exchange chromatography on a Protein-Pack Q 8HR column (Waters) using an HPLC apparatus (Waters 600) equipped with a photodiode array detector. The column was equilibrated in 20 mM sodium acetate buffer pH 5 at a ow 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 proles, 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 ( = 3.6 104 cm1 mol1 l) as reported by Fukushima and Kirk [22]. The assay reaction mixture contained 1 mmol l1 ABTS in 50 mmol l1 citratephosphate buffer at pH 5 and a suitable amount of enzyme. Enzyme activity was expressed in units, U, dened as mol of ABTS oxidized per min. 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 Pite a, 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. One hundred grams pulp were homogenized in 2 l water using a 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 410 kPa). 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 specied), in a glass basin containing the monomeric compounds dissolved in 75 ml 0.2 M citratephosphate buffer at pH 5 under constant shaking (100 rpm) at 50 C. Laccase was added at 15 U/g of paper whereas the concentration of phenol compounds was function of their solubility: caffeic, gallic, p-hydroxybenzoic acids and dopamine were used 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 handsheets were washed three times with 100 ml of distilled water for 10 min, then dried and partially sterilised at 90 C for 1 h.

2.4. Oligomers preparation by laccase treatment

Isoeugenol, p-hydroxybenzoic acid and caffeic acid oligomers were dissolved at 1 mM concentration in 250 ml citratephosphate 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 ultraltrated on 1 kDa molecular cut-off membrane (Amicon YM2, Danvers, MA, USA), using three volumes of 50 mM citratephosphate buffer pH 5 to eliminate the unreacted monomers. The ux during the ultraltration was approximately 0.3 ml/min. The retentate containing the oligomers was rst frozen and then lyophilized at 80 C. The oligomers were maintained at 20 C until use.

2.8. Determination of antibacterial activity of handsheet treated samples

A modied 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 at 100 rpm) with the exception of B. subtilis that was grown at 30 C. The bacteria pre-inocula were diluted with NB medium (NB 25% or NB 12.5%) and 200 l aliquots (containing 103 CFU) were used to inoculate handsheet specimens (2.5 cm 2.5 cm) 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 approximately 15 ml of sterile water to avoid the drying of the paper specimens during incubation. The growth medium (NB used to dilute and 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

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-

G. Elegir et al. / Enzyme and Microbial Technology 43 (2008) 8492 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 ), and the other two to determine the number of cells at the end of the antibacterial test (CFU T24 ). Immediately after inoculation the rst specimen was extracted with 50 ml of neutralising solution to recover inoculated bacteria (CFU T0 determination), while the petri dishes containing the other two inoculated specimens were incubated overnight at 37 C for all bacteria with the exception of B. subtilis (30 C). The neutralising solution had the following composition: 3 g/l l--phosphatydilcoline; 5 g/l sodium thiosulfate; 1 g/l l-histidine; 30 g/l Tween 80; 10 ml/l pH 7 buffer (34 g/l KH2 PO4 ), the nal 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 neutralising solutions were determined by serial dilution plated by inclusion in PCA medium. To evaluate the antibacterial efcacy of the treated samples, the CFU T24 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 CFU for all tests, to claim a bactericidal effect the treated samples should reach a log CFU T24 value of 1.

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reported in literature by Granata and Argyropoulos [25] and Saake et al. [26]. The 31 P spectra were recorded using a Varian Mercury 400 MHz instrument at 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 (100 l) was then added, together with an internal 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 2 102 mmol/g, while the maximum standard error was 1 102 mmol/g.

2.12.

13

C NMR

1D 13 C spectra of acetylated isoeugenol oligomers were recorded using a Varian Mercury 400 MHz instrument at 308 K. The chemical shifts were referred to the solvent signal at 39.5 ppm. Relaxation delay of 10 s was used between the scans. Line broadening of 25 Hz was applied to FIDs before Fourier 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 laccase polymerization, determined by SEC analysis, is reported in Table 1. According to these results a higher average molecular 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 polydispersity 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 corresponding monomers was monitored for 24 h, their duplication times (td ) 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 td 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 td value up to 2.3 h whereas a complete growth inhibition was detected in the presence of the CA oligomer. 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 4934 2596 1.9 p-hydroxybenzoic acid 1857 1101 1.7 Isoeugenol 3652 1530 2.4

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 bres. 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, bres were drained and dispersed in 450 ml of 0.001 M NaCl. Titration was carried out with NaOH 0.1 M, 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 sufcient 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 set at a wavelength of 280 nm. The GP-column was an Agilent PL 3 m MIXED gel E MW 220400 W. The acetylated lignin samples were dissolved in tetrahydrofuran (THF), this solvent was also used as a mobile phase and the ow rate was 0.8 ml min1 . Linear polystyrene standards with molecular weights between 162 and 115,000 g mol1 were used to estimate the molecular weight of the samples. The polystyrenecalibration 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.

31

P NMR analysis

Resonance analysis of phosphorus (31 P 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

Mw (Da) Mn (Da) Mw /Mn

Data were obtained by size exclusion chromatography (SEC) performed in tetrahydrofuran.

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Table 2 Comparison of bacterial growth (td ) in the presence of phenol monomers and oligomers obtained by laccase polymerization Duplication time, td (h) Staphylococcus aureus Control cellsa Control laccaseb Caffeic acid Caffeic acid oligomer Isoeugenol Isoeugenol oligomer 0.65 0.70 0.80 No growth 1.00 2.30 Escherichia coli 0.52 0.57 0.57 No growth Not determined 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.

The molecular structure of isoeugenol and CA oligomer were further investigated by FTIR and 13 C NMR/31 P NMR studies. The acid groups content in the CA oligomer measured by 31 P 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 prole compared to the monomer. The benzene ring ngerprints were different in CA and CA oligomer, the absorption peaks between 1450 and 1600 cm1 , associated with the aromatic ring C C stretching vibration bands, were still present, but the vibration bands of the carboxylic group in the oligomer appeared at 1700 cm1 instead of 1660 cm1 . Moreover, the absorption peak at 1274 cm1 attributed to the CO stretching vibration bands was not anymore present in the spectra of the oligomer. These results along 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 CC 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 linkages. Phenol hydroxyl decreased during polymerization due to phenoxy radicals coupling and O4 intermonomeric bonds were predominant. A signicant amount of intermonomeric 5 bonds were also detected. Based on these preliminary results several phenol compounds known for their antimicrobial activity were used with laccase to covalently bind these monomer/oligomer structures onto the bre 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 efcient than pulp bulk treatments to impart antibacterial activity to bres (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 activity of paper samples. 3.1. Antibacterial bres based on laccase grafting of aromatic acids Antibacterial activity of LASP treated handsheets was initially tested on S. aureus (Fig. 3). Untreated handsheets supported a signicant bacterial proliferation after 24 h contact 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 laccase demonstrated a slight bacteriostatic activity with HBA. In contrast, 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 400 U of enzyme/g of caffeic acid.

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Fig. 3. Antibacterial activity of different LASP treated papers on Staphylococcus 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 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 only with phenolic acids; ( ) LASP treated samples. Abbreviations: CA, caffeic acid; 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 function of HBA concentration in the reaction media, is reported in Fig. 4. For the paper samples prepared in the presence of laccase, the results clearly show that increasing the concentration of HBA the antibacterial activity on E. coli is enhanced, reaching a signicant 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

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 conrmed the absence of activity at relatively low concentrations (46 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 signicant 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 inuence of reaction time on grafting and antibacterial effect was further investigated. The grafting efciency of aromatic acids was measured by titration of the acid groups in the bres before and after laccase reaction (15 U/g). In the presence of CA and HBA the acid group content was increased from 84.2 mol/g (untreated kraft bres) up to 120.6 and 136.0 mol/g, respectively. Increasing the time up to 24 h or the laccase concentration up to 60 U/g did not result in any signicant improvement of the grafting. In Fig. 5 the antibacterial activity of CA/LASP treated handsheets 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 signicant 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 bres with this compound. 3.2. Antibacterial bres 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. 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 reacting 15 U/g laccase in the presence of HBA concentrations ranging from 4 to 36 mM. Symbols: ( ) untreated control; ( ) controls treated only with HBA; ( ) LASP treated samples.

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 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 CA at 4 mM. Symbols: ( ) untreated control; ( ) controls treated only with CA; ( ) LASP treated samples.

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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 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 reacting 15 U/g laccase in the presence of various essential oils components at 4 mM. Symbols: ( ) untreated control; ( ) controls treated only with essential oils; ( ) LASP treated samples.

Fig. 8. Antibacterial activity of DOPA/LASP treated papers on different bacteria. 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 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 DOPA at 60 mM. Symbols: ( ) untreated control; ( ) controls treated only with DOPA; ( ) LASP treated samples. Abbreviations: DOPA, dopamine.

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 signicant bacteriostatic effect, whereas thymol demonstrated a bacteriostatic effect only in the absence of laccase. The antibacterial efcacy 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 signicant antibacterial activity only against Gram positive bacteria with the exception of

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 bres based on laccase grafting of an aromatic amine A preliminary investigation on the use of aromatic amines in the LASP is reported in Fig. 8. In this case 60 mM dopamine concentration was used during an overnight reaction with laccase. As can be observed, dopamine treated handsheets exerted a signicant 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 particular bactericidal activity was detected on the Gram positive spore forming B. subtilis and on Gram negative E. coli, while on K. pneumoniae only a signicant 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 efcacy was tested comparing nine different independent 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, ranging from 2.2 to 3.0 (data not shown) and corresponding to an antibacterial effect of 3.93.1 log reduction. A low variability of the data within a single treatment was also detected indicating 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.

Fig. 7. Antibacterial activity of isoeugenol/LASP treated papers on different bacteria. 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 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 isoeugenol at 0.4 mM. Symbols: ( ) untreated control; ( ) LASP treated samples.

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4. Discussion In the last few years it has been shown by several researchers that laccase can be used to graft new functional groups onto different type of bres upgrading and even changing bre properties [8,9]. Among the potential applications of laccase grafting, the development of antimicrobial bres has received little attention. In this study, laccase was used to graft several aromatic antimicrobial additives bearing different functional groups onto unbleached kraft bres. In literature the antimicrobial properties of these compounds were reported in several studies [1119], however, in order to develop a laccase based antimicrobial process (LASP) two main issues must be rstly considered: the reactivity towards laccase and the maintenance of antimicrobial properties of the grafted compounds. Moreover, also the assessment conditions should be carefully considered, in our work the adopted testing parameters were chosen to mimic as close as possible the conditions to which bres products will be exposed during storage (contact between bacteria and wet bres in the presence of relatively low nutrient concentrations avoiding excess water). In general, also taking into account the reproducibility data obtained with HBA/LASP, the efcacy of the developed laccase grafting procedure to impart antimicrobial activity to paper samples was proved. However, in some cases a high variability was registered when testing duplicates of the same treated paper sample, this could be due to both: inhomogeneity of the paper handsheet treatment and/or variability in the contact conditions among bacteria and bres during antibacterial test. In few cases also control handsheets paper treated with chemicals but without laccase showed antibacterial activity probably due to residues of active substances not covalently bound on the bres surface and not sufciently removed during washings. The antibacterial mechanism of phenols is generally associated with the presence of hydroxyls and delocalisation of the electrons on their structure [18,19]. Furthermore, the branched nature of antimicrobial compounds has been reported to contribute to their activity [28]. Our experiments performed in liquid medium clearly showed that the oligomers had a much higher antibacterial activity than the corresponding monomers. The analysed isoeugenol and caffeic acid oligomers had a lower hydroxyl content but a higher activity than the corresponding monomers, suggesting that several mechanisms besides the presence of this functional group might be responsible of antibacterial activity. The higher electron delocalisation, the branched nature of the surface oligomer and the higher content of carboxyl groups might be claimed to explain the higher antibacterial activity displayed by oligomers. In this work the results obtained with LASP treated handsheet paper demonstrated that the antibacterial properties of the phenol derivatives were maintained after their grafting onto the bres and probably increased by laccase polymerization. On the other hand, from the obtained data it cannot be ruled out that the antibacterial effect displayed by LASP treated paper is simply due to monomers grafting onto the bre surface. Unfortunately, due to the structural similarity of these compounds with lignin, it was not possible to clarify the grafting mechanism and further tests are needed to investigate this issue. As a consequence, differ-

ent strategies of grafting could be also envisaged to optimize the system and/or investigate grafting mechanism, i.e. by selecting the reaction conditions, laccase could be used to build up a grafted hyper-branched oligomer possibly increasing antimicrobial activity either separately or directly onto the bre surface. According to our data the antibacterial properties of LASP treated bres were function of the grafted phenolic structure. More specically regarding aromatic acids, CA and HBA were more effective in comparison to GA, showing signicant reductions of the Gram positive S. aureus load at low concentration. HBA/LASP also showed a signicant antibacterial effect towards the Gram negative E. coli, but at higher concentrations. CA displayed the advantage of being more reactive with laccase (data not shown) than HBA, thus allowing a shorter grafting reaction time (Fig. 5), but due to its lower solubility it could only be used at relatively low concentrations. Essential oils are another very interesting class of natural products showing antibacterial properties, among them eugenol has been widely applied in dental eld [29], thymol and carvacrol have been shown to display activity against food-borne pathogens such as Bacillus cereus [19]. In our experiments isoeugenol/LASP showed encouraging results, even if, among the different microorganisms tested, the antibacterial activity occurred only with Gram positive bacteria including the spore forming B. subtilis. On the contrary, eugenol/LASP was less effective and thymol/LASP did not prevent bacterial growth. These data are consistent with literature references where the highest activity of essential oils versus Gram positive bacteria is often reported although many exceptions suggest that it cannot be taken as a general rule [2,16,29]. The overall results obtained in our work suggest that Gram positive bacteria are more sensible towards LASP modied bres. Laccase initiated grafting and/or polymerization of antibacterial aromatic structures increases the efcacy of these antibacterial substances and might be pursued as a valid methodology to build up covalently bound bio-active surfaces meanwhile decreasing the amount of chemicals and possibly the related migration issues. Although laccase antibacterial surface process (LASP) will need a deeper investigation, potential applications are envisaged in hygiene paper products and active packaging to improve food shelf life. In future perspective the synergism of different chemical structures with respect to their antimicrobial activity on different classes of bacteria, and the use of natural extracts instead of puried compounds will be taken into account. Acknowledgements The authors thank the European Commision (SustainPack IP-500311-2), the Italian Minister of Economy (MEF-Fondo di Rotazione nazionale) and Comieco (Italian Consortium for the Recovery and Recycling of Cellulose-based Packaging) for their nancial support. References
[1] Cha DS, Chinnan MS. Biopolymer-based antimicrobial packaging: a review. Crit Rev Food Sci Nutr 2004;44:22337.

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G. Elegir et al. / Enzyme and Microbial Technology 43 (2008) 8492 microbial metabolites of berry-derived phenolic compounds and organic acids. J Agric Food Chem 2007;55:390512. Burt S. Essential oils: antibacterial properties and potential applications in fooda review. Int J Food Microbiol 2004;94:22353. Banes-Marshall L, Cawley P, Phillips CA. In vitro activity of Melaleuca alternifolia (tea tree) oil against bacterial and Candida spp. isolates from clinical specimens. Br J Biomed Sci 2001;58:13945. Knobloch K, Pauli A, Iberl B, Weigand H, Weis N. Antibacterial and antifungal properties of essential oil components. J Essent Oil Res 1989;1:11928. Ultee A, Bennik MHJ, Moezelaar R. The phenolic hydroxyl group of carvacol is essential for action against the food-borne pathogen Bacillis cereus. Appl Environ Microbiol 2002;68:15618. Harkental M, Reichling J, Geiss HK, Saller R. Comparative study on the in vitro antibacterial activity of Australian tea tree oil, cajuput oil, niaouli oil, manuka oil, kanuka oil, and eucalyptus oil. Pharmazie 1999;54:4603. Galhaup C, Wagner H, Hinterstoisser B, Haltrich D. Increased production of laccase by wood-degrading basidiomycete Trametes pubescens. Enzyme Microb Technol 2002;30:52936. Fukushima Y, Kirk TK. Laccase component of the Ceriporiopsis subvermispora lignin degrading system. Appl Environ Microbiol 1995;61:8726. Katz S, Beatson RP, Scallan AM. The determination of strong and weak acidic groups in sulte pulps. Svensk Papperstidning 1984;87:4853. Himmel ME, Tatsumoto K, Oh KK, Grohmann K, Johnson DK, Li Chum H. Molecular weight distribution of aspen lignins estimated by universal calibration. In: Glasser WG, Sarkanen S, editors. Lignin properties materials, vol. 6. Washington, DC: American Chemical Society; 1989. p. 8299. Granata A, Argyropoulos DS. 2-Chloro-4,4,5,5-tetramethyl-1,3,2dioxaphospholane a reagent for the accurate determination of the uncondensed and condensed phenolic moieties in lignins. J Agric Food Chem 1995;43:153844. Saake B, Argyropoulos DS, Beinhoff O, Faix O. A comparison of lignin polymer models (DHPs) and lignins by P-31 NMR spectroscopy. Phytochemistry 1996;43:499507. Xu P, Uyama H, Whitten JE, Kobayashi S, Kaplan DL. Peroxidasecatalyzed in situ polymerization of surface oriented caffeic acid. J Am Chem Soc 2005;127:1174553. Chen CZ, Cooper SL. Interaction between dendrimer biocides and bacterial membranes. Biomaterials 2002;16:335968. Shapiro S, Meier A, Guggenheim B. The antimicrobial activity of essential oils and essential oil components towards oral bacteria. Oral Microb Immunol 1994;9:2028.

[2] Kalemba D, Kunicka A. Antibacterial and antifungal properties of essential oils. Curr Med Chem 2003;19:81329. [3] Leonowicz A, Cho NS, Luterek J, Wilkolazka A, Wasilewska MW, Matuszewska A, et al. Fungal laccase: properties and activity on lignin. J Basic Microbiol 2001;41:185227. [4] Wong KKY, Richardson JD, Manseld SD. Enzymatic treatment of mechanical pulp for improving papermaking properties. Biotechnol Prog 2000;16:10259. [5] Felby C, Hassingboe J, Lund M. Pilote-scale production of breboard made by laccase oxidized wood bers: board properties and evidence for crosslinking of lignin. Enzyme Microb Technol 2002;31:73641. [6] Gr onqvist S, Buchert J, Rantanen K, Viikari L, Suurn akki A. Activity of laccase on unbleached and bleached thermomechanical pulp. Enzyme Microb Technol 2003;32:43945. [7] Kurek B, Martinez-Inigo MJ, Artaud I, Hames BR, Lequart C, Monties B. Structural features of lignin determining its biodegradation by oxidative enzymes and related systems. Polym Degrad Stabil 1998;59:359 64. [8] Chandra RP, Lehtonen LK, Ragauskas AJ. Modication of high lignin content kraft pulps with laccase to improve paper strength properties. 1. Laccase treatement in the presence of gallic acid. Biotechnol Prog 2004;20:25561. [9] Buchert J, Gr onqvist S, Mikkonen H, Oksanen T, Peltonen S, Suurn akki A, Viikari L. Process for producing a brous product. European Patent WO2005061790; 2005. [10] Chandra RP, Ragauskas AJ. Evaluating laccase facilitated coupling of phenolic acids to high-kappa kraft pulps. Enzyme Microb Technol 2002;30:85561. [11] Nohynek LJ, Alakomi HL, Kahkonen MP, Heinonen M, Helander IM, Oksman-Caldentey KM, et al. Berry phenolics: antimicrobial properties and mechanisms of action against severe human pathogens. Nutr Cancer 2006;54:1832. [12] Kubo I, Fujita K, Nihei K, Masuoka N. Non-antibiotic antibacterial activity of dodecyl gallate. Bioorg Med Chem 2003;11:57380. [13] Rodr guez Vaquero MJ, Alberto MR, Manca de Nadra MC. Inuence of phenolic compounds from wines on the growth of Listeria monocytogenes. Food Control 2007;18:58793. [14] Almeida AA, Farah A, Sylva DA, Nunan EA, Gloria MB. Antibacterial activity of coffee extracts and selected coffee chemical compounds against enterobacteria. J Agric Food Chem 2006;54:873843. [15] Alakomi HL, Puupponen-Pimia R, Aura AM, Helander IM, Nohynek L, Oksam-Caldentey KM, et al. Weakening of Salmonella with selected

[16] [17]

[18]

[19]

[20]

[21]

[22] [23] [24]

[25]

[26]

[27]

[28] [29]