Tyrosinase-catalysed-grafting-of-food-grade-gallates-to-chitosan2008.pdf

PACKAGING TECHNOLOGY AND SCIENCEPackag. Technol. Sci. (2008)Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/pts.813

Tyrosinase-catalysed Grafting of Food-grade Gallates to Chitosan: Surface Properties of Novel Functional Coatings

By Jari Vartiainen,* Marjaana Rtt, Raija Lantto, Kalle Nttinen and Eero HurmeVTT Technical Research Centre of Finland, PO Box 1000, FI-02044 VTT, Finland

Plasma-activated biaxially oriented polypropylene (BOPP) fi lms and paper substrate have been coated with functional chitosan solutions. Plasma treatment increased the amount of surface peroxide groups and carboxyl groups on the BOPP fi lms. As a result of plasma activation, the surface energy increased from 30 to 50 dynes/cm. The enzyme tyrosinase catalysed the grafting of octyl gallate and dodecyl gallate to amino groups of chitosan polysaccharide. Resulting coatings exhibited strong antimicrobial activity against Gram-positive Staphylococcus aureus and Gram-negative Listeria innocua. After 24 h of incubation, a total reduction in both bacteria cell numbers varied between >4.9 and 1.4 logarithmic units. Grafted dodecyl gallate and octyl gallate at pH 6 were found to have the lowest reduction values of

J. VARTIAINEN ET AL.Packaging Technologyand Science

Copyright 2008 John Wiley & Sons, Ltd. Packag. Technol. Sci. (2008)DOI: 10.1002/pts

storage.1 Biobased packaging materials are defi ned as materials from renewable sources. There is increasing interest in functional packaging materi-als that utilize biobased components or layers. The extended use of waste by-products of agriculture and the food industry will be of particular value in the future.

Gallic acid is an organic acid found in oak bark, tea leaves and many other plants. Gallates are syn-thesized by the esterifi cation of gallic acid. Propyl (E310), octyl (E311) and dodecyl (E312) gallates are used as antioxidant additives in foods, cosmetics and medicinal preparations (Figure 1). Octyl gallate has been shown to exhibit strong antifungal activ-ity against Saccharomyces cerevisiae, Zygosaccharo-myces bailii, Candida albicans and Aspergillus niger. The activity was not infl uenced by pH variations. The primary activity of octyl gallate comes from its ability to act as a non-ionic surfactant without need to enter the cell. Gram-positive bacteria such as Bacillus subtilis, Micrococcus luteus and Staphylococ-cus aureus have been shown to be susceptible to gallates while Gram-negative bacteria with the exception of Salmonella choleraesuis are mostly resistant. Octyl and dodecyl gallates are clearly more effi cient as compared to propyl gallate.2,3 Commercially unavailable gallates with varying chain lengths have been synthesized and their antimicrobial properties against methicillin-resis-tant S. aureus (MRSA) were evaluated. The length of the alkyl chain was found to be associated with antimicrobial effi ciency, and the optimum chain lengths were C9 (nonyl gallate) and C10 (decyl gallate).4

Tyrosinase is an enzyme that is known to catalyse the oxidation of mono- and diphenols and is wide-spread in plants and animals. Also some short-

chain gallates have been oxidized by tyrosinase.57 Although these oxidation rates initially were slow, they were signifi cantly increased by adding L-3,4-dihydroxyphenylalanine (L-DOPA) as a booster. Tyrosinase catalyses the oxidation of phenols to electrophilic ortho-quinones which are able to freely diffuse and covalently bind to, for example, the nucleophilic amine groups of chito-san. Chitosan is a polysaccharide prepared by N-deacetylation of chitin, the second most abundant natural biopolymer after cellulose. Chitosan is an edible and biodegradable material, which also has antimicrobial activity against different groups of micro-organisms, bacteria, yeasts and moulds.8 The activity of chitosan is mainly based on its amino groups which are positively charged below pH 6.

Tyrosinase-catalysed generation of quinones and their reactivity towards chitosan have been extensively reviewed.9,10 Unlike cellulose or chitin, the high content of primary amino groups in chi-tosan makes it an excellent target for enzymatic modifi cation by tyrosinase. Tyrosinase has been used for the enzymatic grafting of phenolic moi-eties11 and peptides derived from casein and green fl uorescent protein12,13 onto chitosan. It is also reported to catalyse the oxidation of tyrosine resi-dues of silk fi broin and sericin peptides which were further coupled with chitosan resulting in bioconjugates with improved properties.1416 Furthermore, gelatin-chitosan gels and chitosan-polyhydroxystyrene bioconjugates with unique properties have been synthesized by tyrosinase.17,18 Tyrosinase has been immobilized onto chitosan fi lms aimed at various sensor applications.19,20 Tyrosinase-containing chitosan has been exploited in selective phenol removal.2125 It may also have

Figure 1. Structures of food-grade gallates.

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TYROSINASE-CATALYSED GRAFTING GALLATES TO CHITOSAN Packaging Technologyand Science

potential to be used in detection of phenol vapours, such as volatile p-cresol.26 Tyrosinase from Tricho-derma reesei fungus has been recently reported to react with complex compounds such as proteins and proteinaceous fi bres more effi ciently than the well-known and commercially available tyrosinase from a common mushroom Agaricus bisporus.27

Research activity on both tyrosinase and chito-san has increased recently and their promising properties for biotechnological applications in food, packaging, pharmaceutical and environmen-tal fi elds have been acknowledged. Tyrosinase-catalysed grafting of gallates to chitosan may provide a safe way to enlarge environmentally friendly polymers with useful new properties. The aim of the present work was to investigate the effects of grafting on the packaging-related surface properties of chitosan coatings.

MATERIALS AND METHODS

Chitosan, medium molecular weight, was obtained from Aldrich Chemical Company, Inc. (Milwau-kee, WI, USA). T. reesei tyrosinase TYR2 was char-acterized and produced at VTT, Finland as previously described.28 Dodecyl gallate (DOGA) was from Merck (8.20598.0100, Hohenbrunn, Germany) and octyl gallate (OGA) from Lancaster (6081, Lancashire, UK). Biaxially oriented polypro-pylene (BOPP) standard commercial-grade fi lms were obtained from Radic (Radil lb transparent BOPP 30 mm, San Giorgio di Nogaro, Italy).

Plasma activation

Plasma activation of BOPP fi lms was carried out in atmospheric conditions using Plasmatreat (Steinhagen, Germany) FG1002 impulse discharge system with RD 1004 plasmanozzle. Treatment parameters were as follows: speed 720 mm/min, power 300 W, 2950 rpm, distance 13 mm. Pressur-ized air was used as treatment gas.

Surface densities of peroxides and carboxyl groups

Peroxides produced on the surface of plasma-acti-vated BOPP fi lms were determined by the iodide

method spectrophotometrically. This method uti-lizes an oxidation of sodium iodide by peroxides in the presence of ferric chloride. The plasma-treated fi lms (1 3 cm2) were placed in 7 ml of isopropyl alcohol solution containing 60 mg of sodium iodide and 1 ppm of ferric chloride and kept at 60C for 10 min. After addition of water to stop the reaction, the oxidized iodine was measured as triiodide anion from the absorbance of the solution at 360 nm with the molar absorptivity of 2.3 104 l/mol cm. Surface densities of carboxyl groups were evalu-ated from the uptake of basic dye. For determina-tion of carboxyl groups, fi lm samples (0.5 dm2) were immersed in 20 ml of 0.01 g/ml Toluidine Blue O (Merck, Darmstadt, Germany) of pH 10 at room temperature for 5 h. Non-complexed dye was removed with distilled water and 1 mM NaOH and desorption of dye molecules complexed to the car-boxyl groups on the fi lm surface was conducted with 50% acetic acid solution. The dye concentra-tion was determined at 633 nm by using a spectro-photometer and calculated from the calibration curve. The surface densities of functional groups were calculated assuming that Toluidine Blue O was complexed to equivalent moles of carboxylic groups.

Tyrosinase treatment

Tyrosinase-treated chitosan solutions for coating purposes were made by fi rst preparing a 2% chi-tosan solution in 1% acetic acid (pH 4). Chitosan powder was mixed with acetic acid, after which the solution was let to dissolve for 2 days at room temperature. Filtration through metal sieve and degassing was necessary to minimize the amounts of undissolved impurities and air bubbles. DOGA and OGA were dissolved in ethanol and added to chitosan solutions. The fi nal concentration of gal-lates was adjusted to 0.2%. Concentration of ethanol in fi nal coating solutions was kept below 10%. pH was adjusted using 1 M NaOH. Finally, tyrosinase (500 nkat/g chitosan) was added under rigorous mixing.

Coatings

Coating solutions were fi rst modifi ed and then applied onto copy paper and plasma-activated

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BOPP fi lm using RK Control laboratory coater (model K101, RK Print Coat Instruments Ltd., Lit-lington, Royston, Herts, UK). Drying of the coated samples was performed in an air-circulation labo-ratory oven at 50C. Three coating layers were applied onto each sample using the standard coating bar no. 6 (wet fi lm deposit of 60 mm), and dried for 10 min after each single layer. Total amount of the dried coating was approximately 3 g/m2. Coated samples were further dried for 1 day at room temperature and stored at 4C until tested.

Viscosity

Prior to viscosity measurements, the dried chitosan samples were immersed in 1% acetic acid and let to dissolve at room temperature for 5 days. Viscosity of chitosan solutions was measured using Brook-fi eld (Middleboro, MA, USA) Model DV-III Rhe-ometer at 23C using spindel DIN-87, model LV and rotation speed of 25 rpm.

Colour

Hunter b-values were measured using a colorim-eter (CR-200 Minolta Chroma Meter, Minolta Camera Co., Osaka, Japan). The b-value indicates the colour direction: +b (yellow) and b (blue). Colour values were determined randomly at three different positions using two individually pre-pared solutions, thus the values were averaged from totally six replicated readings. 10 ml of each solution was poured onto Petri dishes with a diam-eter of 5 cm and measured on the surface of stan-dard white calibrating plate. The relative b-values of modifi ed solutions were compared to reference b-value of pure chitosan.

Scanning electron microscopy (SEM)

SEM JEOL JSM-T 100 with accelerating voltage control of 15 kV was used to investigate the cross- section of chitosan-coated BOPP fi lms.

Water contact angle

Water contact angles of the coated fi lms were mea-sured using CAM-200 equipment (KSV Instru-

ments, Helsinki, Finland) in test conditions of 23C and 50% relative humidity (RH). Contact angle values were measured after incubation for 2 s.

Water vapour transmission

Water vapour transmission rates were determined gravimetrically using a modifi ed ASTM E-96 pro-cedure. Circular samples ( 6.6 cm) were cut from the chitosan-coated paper and mounted tightly on the mouth area of an aluminium dish (H.A. Bchel V/H, A.v.d. Korput, Baarn-Holland 45M-141) con-taining water. Dishes were stored in climate room in test conditions of 23C and 50% RH and weighed periodically until a constant rate of weight reduc-tion was attained. Weightings were used to deter-mine the amount of moisture transferred through the coated paper from the cup towards lower humidity in climate room. The RH difference (the humidity gradient) during the test was 50%.

Oxygen transmission

Oxygen transmission measurements were per-formed with Ox-Tran 2/20 Oxygen Transmission Rate System (Mocon, Modern Controls, Inc., Min-neapolis, MN, USA) using the methods described in standards ASTM D3985 and F1927. Tests were carried out at 23C and 0, 50 and 80% RH using 100% oxygen as test gas.

Antimicrobial activity

Antimicrobial activity was determined as described in standard JIS Z 2801 Antimicrobial products test for antimicrobial activity and effi cacy. Bacterial suspensions of Staphylococcus aureus and Listeria innocua containing approximately 1 106 CFU/ml of test bacteria were placed on the surface of 5 5 cm2 sized chitosan-coated BOPP fi lms, covered with a plastic foil and incubated for 24 h at 35C. After incubation the bacteria were washed from the samples and the number of viable bacteria was measured by plating. The number of viable bacte-ria eluted from the antimicrobial samples was com-pared to that eluted from the uncoated reference BOPP fi lm.



Migration test

Migration tests were carried out as described in European prestandard ENV 1186-3 Materials and articles in contact with foodstuffs Plastics Part 3: Test methods for overall migration into aqueous food simulants by total immersion. 95% ethanol was used as food simulant in test conditions of 24 h at 40C. As both gallates are insoluble in water and freely soluble in ethanol, 95% ethanol was chosen as a food simulant. It can be used as alcoholic food simulant or alternative fatty food simulant. Test was performed by immersing test specimens of 1 dm2 in food simulant, after which the simulant was evaporated to dryness and the overall mass of the residue was determined. The analytical tolerance of the test was 1 mg/dm2.

RESULTS AND DISCUSSION

BOPP fi lms without additional surface activation are too hydrophobic for most water-based coating solutions. Incomplete wettability of the hydropho-bic substrate results in mottle coatings with poor surface properties. Typically, this is not a problem with paper-based substrates which are porous allowing the coating solution to penetrate into surface layer of the fi bre matrix. For increasing the

hydrophilicity of the BOPP fi lms, the plasma acti-vation in atmospheric conditions was carried out using lab-scale plasma treatment. As a result of plasma activation, the surface energy increased from 30 to 50 dynes/cm which was adequate for good wettability. Plasma activation increased the amount of surface peroxide groups from 3.2 1010 to 1.6 109 mol/cm2 and the number of carboxyl groups from 1.2 1010 to 5.4 1010 mol/cm2. Adding functional groups onto surface enables not only the better wettability but also provides possi-ble target sites for suitable linkable biomolecules.

Prior to viscosity measurements the dried chito-san samples were immersed in 1% acetic acid. Chi-tosan without any additives was readily dissolved and formed completely clear and transparent solu-tion. Chitosan mixed with DOGA formed white opaque solution whereas the solution containing OGA was white but transparent. Chitosan contain-ing both gallates and tyrosinase formed yellowish solutions. Tyrosinase-initiated quinone formation and subsequent non-enzymatic reaction of the qui-nones lead to pigmented compounds. Due to oxidation of gallates during the tyrosinase treat-ments, the chitosan coatings turned yellowish. As increased b-values indicated, the tyrosinase-cata-lysed grafting was more powerful with OGA. With both gallates, the enzymatic grafting was increased at pH 6 (Figure 2). As reported previously, shorter

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Figure 2. +b-values (yellowness) of modifi ed chitosan was measured using CR-200 Minolta Chroma Meter. The relative b-values of modifi ed

solutions were compared to reference b-value of pure chitosan.



chain gallates such as octyl gallate can be easily oxidized by tyrosinase yielding yellowish oxida-tion products whereas long-chain gallates such as dodecyl gallate are not that sensitive. The long hydrophobic chains may interact with copper active site in tyrosinase resulting in lower activ-ity.2931

Tyrosinase is proposed to oxidize the phenolic structures of gallates to the corresponding qui-nones. The functional amino groups of chitosan may further react with gallate quinones through the Schiff-base (1) or Michael-type (2) reaction mechanisms18 as shown in Figure 3.

Chitosan with OGA and tyrosinase formed fl oc-culated solutions, especially at pH 6 indicating the

presence of grafted or cross-linked insoluble mate-rial. Reaction between tyrosinase and DOGA was not as evident although at pH 6 the solution was slightly fl occulated. Due to partial fl occulation, the viscosity of all solutions was not measurable (Table 1). Phenolic compound (p-cresol) and tyrosinase have been previously added to chitosan solutions (pH 6) and further incubated overnight at room temperature. The viscosity measurements indi-cated that the chitosan solutions had been con-verted into a cross-linked gel.32

As the SEM cross-section picture indicates (Figure 4), the contact between chitosan coatings and BOPP substrate was relatively strong and the thickness of the chitosan layer was below 2 mm.

Figure 3. Proposed reactions in tyrosinase-catalysed grafting of gallates to chitosan.

Table 1. Viscosity of modifi ed chitosan was measured at 23C using rotation speed of 25 rpm

Sample Viscosity, mPas

Chitosan 89Chitosan + DOGA 106Chitosan + OGA 108Chitosan + DOGA + tyrosinase (pH 4) 110Chitosan + OGA + tyrosinase (pH 4) Partly fl occulatedChitosan + DOGA + tyrosinase (pH 6) Partly fl occulatedChitosan + OGA + tyrosinase (pH 6) Partly fl occulated

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Chitosan has a high natural affi nity for water as can be seen in Figure 5. Its hydrophobicity was enhanced using both free and grafted gallates. Tyrosinase-catalysed grafting at pH 6 slightly increased hydrophilicity as compared to pH 4 (Figure 6). As demonstrated before, the enhanced cross-linking increased the hydrophilicity of chito-san33,34 and it can be further modifi ed by varying the reaction conditions and gallate ester function-ality.35 In our study, DOGA-containing chitosan coatings were more hydrophobic as compared to OGA.

Oxygen transmission rates of all chitosan-coated BOPP fi lms were strongly dependent on humidity

(Figure 7). In dry conditions, the transmission rate of 2 cm3/(m2 24 h) was obtained with both chito-san (without gallates) and DOGA grafted chitosan (pH 6) coatings. Signifi cant increase in transmis-sion was found with increasing humidity, thus 50 cm3/(m2 24 h) transmission was measured for chitosan (without gallates) coatings in 50% RH and 750 cm3/(m2 24 h) transmission in 80% RH. Hydro-phobic gallates with or without tyrosinase did not improve the barrier properties in high humidity. DOGA-containing coatings had slightly better barrier properties as compared to OGA. Tyrosi-nase-catalysed grafting of both gallates to chitosan at pH 6 resulted in lower oxygen transmission in dry conditions but notably higher transmission in 50 and 80% RH. This may be a consequence of the enhanced cross-linking which increased the hydro-philicity and oxygen transmission in high humid-ity. However, all chitosan-coated BOPP fi lms reduced the oxygen transmission as compared to humidity independent value of the reference BOPP fi lm [1250 cm3/(m2 24 h)].

Chitosan coatings did not have major effects on water vapour transmission (Figure 8). The repeated weightings were used to determine the amount of moisture transferred through the chitosan-coated 80 g/m2 copy paper towards lower humidity in climate room. The RH difference (the humidity gradient) during the test was around 50%. Only minor differences were found between the samples. All samples including the reference copy paper

Figure 4. SEM cross-section picture of chitosan-coated plasma-activated BOPP fi lm.

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Figure 5. Water contact angle of chitosan-coated paper.

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without chitosan coatings had water vapour trans-mission rates between 1200 and 1300 g/(m2 24 h). Free gallates slightly decreased the transmission as compared to grafted gallates. DOGA-containing coatings had slightly better barrier properties as compared to OGA. Apparently, the amount of hydrophobic gallates was not adequate or they did not form an even and effective barrier against the transmission of water vapour.

Migration of substances into 95% ethanol was measured using total immersion method. Ethanol

is a good solvent for gallates whereas chitosan and BOPP are not soluble in ethanol. As can be seen in Table 2, the reference BOPP and chitosan-coated BOPP had the lowest migration of 4 mg/dm2. This amount of migrated substances exceeded the quantity of incorporated gallates and tyrosinase into chitosan. As reported previously, the graft copolymers of chitosan may exhibit an improved affi nity for organic solvents, unlike the original chitosan.36 In this case, gallate-grafted chi-tosans may have become slightly soluble in ethanol causing increased migration into used simulant. All migration values were clearly below the limit value of 10 mg/dm2, thus the materials meet the requirements set for the total migration of sub-stances migrated from the packaging materials into foodstuffs stipulated in Directive 2002/72/EC.

Antimicrobial properties were determined as described in standard JIS Z 2801. Activity was expressed using R-value as follows: R = log B

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Figure 6. Water contact angle of gallate-grafted chitosan coatings on plasma-activated BOPP.

Figure 7. Oxygen transmission measured at 23C, 0, 50 and 80% RH.



log C, where B = viable cells on reference after 24 h and C = viable cells on sample after 24 h. Materials were evaluated as having antimicrobial properties if calculated reduction value of R 2 was attained. All chitosan-coated BOPP fi lms were effective against both Gram-positive S. aureus and Gram-negative L. innocua (Tables 3 and 4). After 24 h of incubation, a total reduction of >4.9 logarithmic units in bacteria cell numbers was observed with all chitosan-coated samples excluding the grafted DOGA at pH 6. Grafted DOGA and OGA at pH 6 were found to have the lowest reduction values of



Table 4. Antimicrobial activity of modifi ed chitosan coatings against L . innocua

Coating

Amount of viable bacteria (Log10 CFU)

Log reduction of viable bacteriaReference sample (B) Antimicrobial sample (C)

Chitosan 5.4 4.7Chi/DOGA 5.4 4.7Chi/OGA 5.4 4.7Chi/DOGA/tyr pH 4 5.4 4.7Chi/OGA/tyr pH 4 5.4 4.7Chi/DOGA/tyr pH 6 5.4 4.0 1.4Chi/OGA/tyr pH 6 5.4 4.7

Table 3. Antimicrobial activity of modifi ed chitosan coatings against S. aureus

Coating

Amount of viable bacteria (Log10 CFU)

Log reduction of viable bacteriaReference sample (B) Antimicrobial sample (C)

Chitosan 5.6 4.9Chi/DOGA 5.6 4.9Chi/OGA 5.6 4.9Chi/DOGA/tyr pH 4 5.6 4.9Chi/OGA/tyr pH 4 5.6 4.9Chi/DOGA/tyr pH 6 5.6 3.0 2.6Chi/OGA/tyr pH 6 5.6 2.7 2.9

catalysed grafting of gallates to amino groups of chitosan at pH 6 decreases the amino functionality of chitosan as well as the diffusion of free gallates into contact with bacteria thus limiting the total activity.

CONCLUSIONS

Tyrosinase-catalysed grafting of food-grade gal-lates (E311 and E312) to chitosan was carried out at pH 6. The chitosan/gallate solutions were applied as functional coatings on plasma-activated BOPP fi lms and paper substrate. Free or grafted gallates did not improve the antimicrobial proper-ties against S. aureus or L. innocua. This might be a consequence of strong inherent activity of chitosan which masked the additional effects of gallates. As the strain selection was quite limited, it is reason to believe that positive response from gallates may be better recognizable with other microbes. In addition to oxygen barrier and antimicrobial prop-

erties, the grafted gallates may be exploited as functional additives in food packaging materials with modifi ed surface hydrophobicity or improved adhesion towards other biopolymers.

ACKNOWLEDGEMENTS

We thank Ms Pirjo Hakkarainen, Ms Heli Nyknen and Mr Juha Hokkanen for the technical help. We also thank all project partners as well as Tekes (the Finnish Funding Agency for Technology and Innovation) and VTT Tech-nical Research Centre of Finland for funding this study (funding decision 40409/05). The work was carried out within Plastek-project Surface Modifi cation with Plasma and Corona Techniques.

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