Accepted Manuscript Title: Pectin functionalized with natural fatty acids as antimicrobial agent Author: Enrica Calce Eleonora Mignogna Valeria Bugatti Massimiliano Galdiero Vittoria Vittoria Stefania De Luca PII: DOI: Reference:
S0141-8130(14)00238-4 http://dx.doi.org/doi:10.1016/j.ijbiomac.2014.04.011 BIOMAC 4278
To appear in:
International Journal of Biological Macromolecules
Received date: Revised date: Accepted date:
17-12-2013 5-4-2014 7-4-2014
Please cite this article as: E. Calce, E. Mignogna, V. Bugatti, M. Galdiero, V. Vittoriac, S. De Luca, Pectin functionalized with natural fatty acids as antimicrobial agent, International Journal of Biological Macromolecules (2014), http://dx.doi.org/10.1016/j.ijbiomac.2014.04.011 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Graphical abstract
PecPecLinoleate Oleate
80%
PecPalmitate
Pectin
40%
Antimicrobial activity Escherichia Coli
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Staphilococcus Aureus
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0%
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*Manuscript
Pectin functionalized with natural fatty acids as antimicrobial agent Enrica Calce,a Eleonora Mignogna,b Valeria Bugatti,c Massimiliano Galdiero,b Vittoria Vittoriac and Stefania De Luca*a Institute of Biostructures and Bioimaging, National Research Council, 80134 Naples, Italy.
b
Department of Experimental Medicine, II University of Naples, Via De Crecchio 7, 80138, Naples, Italy
c
Department of Industrial Engineering, University of Salerno, 84084 Fisciano (SA), Italy
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a
Corresponding author. Tel: +39 0812534514; fax: +39 0812536642; e-mail: [email protected]
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Abstract
Several pectin derivatives were prepared by chemical modifications of the polysaccharide with natural fatty
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acids. The obtained biodegradable pectin-based materials, pectin-linoleate, pectin-oleate and pectinpalmitate, were investigated for their antimicrobial activity against several bacterial strains, Staphylococcus
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aureus and Escherichia coli. Good results were obtained for pectin-oleate and pectin-linoleate, which inhibit the growth of the selected microorganisms by 50-70%. They exert the better antimicrobial activity against S. aureus.
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Subsequently, the pectin-oleate and the pectin-linoleate samples were coated on polyethylene films and were assessed for their capacity to capture the oxygen molecules, reducing its penetration into the polymeric support. These results confirmed a possible application of the new materials in the field of active food
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packaging.
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Keywords: chemical modification, fatty acid ester of pectin, antimicrobial activity, oxygen scavenging function, active packaging systems
1. Introduction
During the past decade, an increasing interest has been devoted to the development of antimicrobial
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systems to be used as biocides in various areas. In particular, recyclability and biodegradability are nowadays considered important issues when new products with antimicrobial properties are introduced for various applications in medical, pharmaceutical, agriculture, and packaging fields [13]. Free fatty acids (FFA) can be considered natural products, since they are usually provided by natural resources, like triglycerides or phospholipids, and for this reason can be employed with the great advantage of low environmental impact. Within their broad spectrum of biological activities, free fatty acids are able to kill or inhibit the growth of several pathogenic bacteria [4,5]. Whilst their antibacterial mode of action is still unclear, fatty acids have as prime target the bacterial cell membrane and the various essential processes that occur within and at the membrane. Other
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processes that may contribute to bacterial growth inhibition or death include cell lysis, inhibition of enzyme activity, impairment of nutrient uptake and the generation of toxic peroxidation and autooxidation product [6]. However, the usage of FFA as antibacterial agents suffer of several limitations, since some FFAs have an unpleasant taste, while others can be unstable and also have a tendency to bind nonspecifically to proteins. An alternative route for exploring the commercial potential of FFA as antimicrobials could be the conjugation of them to specific carriers, like
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biodegradable material, in order to modulate their delivery. In this regard, biodegradable polymers, produced from natural, renewable resources, can represent ideal candidates, since they readily
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decompose thus reducing the negative environment impact [7-9]. We have recently developed a synthetic process to chemically modify the pectin from apple via acylation of its alcoholic functions
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with several natural fatty acids [10-12]. The aim was to generate new materials by using renewable resources and limiting the number of chemicals and reaction steps. Indeed, natural polymers suffer
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of low water resistance and poor mechanical properties (tensile strength and elongation to break), and these drawbacks are limiting factors for their use as manufactured materials. The chemical modification of pectin with fatty acids as a matter of fact improved water resistance and barrier
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properties of the polysaccharide, thus generating new bio-based material for novel applications. Due to the introduction of fatty acids, these pectin derivatives can be studied for their antimicrobial
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properties, and eventually considered as coatings to be used for the food packaging. With these ideas in our mind, we decided to prepare several pectin samples functionalized with different fatty acids (palmitic, oleic and linoleic acid) in order to investigate their antimicrobial
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activity against two foodborne phatogens, Staphylococcus aureus (Gram-positive) and Escherichia coli (Gram-negative) [8].
2. Experimental
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2.1 Materials
The apple peel Pectin was purchased from Fluka. It is a powder sample with high molecular weight (30 000-100 000 g/mol) and a high degree of esterification (70-75%) on a dry basis. The fatty acids and all solvents were purchased from Sigma-Aldrich.
2.2 Synthesis of pectin derivatives Synthesis of fatty acid anhydrides was performed as follows. The appropriate fatty acid (10 mmol) was dissolved in dichloromethane (2 mL), the solution was cooled in an ice-water bath and stirred vigorously under argon atmosphere. The dicyclohexylcarbodiimide (5 mmol), previously dissolved in the minimum volume of dichloromethane, was added and stirring was continued at ice bath
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temperature for 2 h. The white solid N,N’-dicyclohexylurea was removed by filtration and the solvent was evaporated in vacuo to give the final anhydride [10]. By using an agate mortar, 30 mg of pectin were manually milled with 30 mg of the appropriate fatty acid anhydride in the presence of K2CO3 (0.1 equiv) and few drops of ethanol to obtain the different pectin-derived materials. Reactions were carried out in a domestic microwave oven and irradiated with microwaves (900 W) for two cycles of 3 minutes each. After cooling at room temperature, the
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final crude product was washed with ethyl acetate. The obtained solid was dissolved in water, and 0.5 N HCl was added to the final solution until a neutral pH was reached. This solution was then
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dialyzed (membrane cut off 6000-8000) for 1 day in Milli-Q water and finally lyophilized to give
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the desired product.
2.3 Characterization
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All modified pectin samples were analyzed by FT-IR spectroscopy. The FT-IR spectra were recorded on a Jasco spectrometer. Samples were ground into a fine powder using an agate mortar before being compressed into KBr discs. The characteristic peaks of IR transmission spectra were
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recorded at a resolution of 4 cm-1 over a wavenumber region of 400-4000 cm-1. The bands relevant for the structural organization are: pectin-linoleate (1): FT-IR (cm-1): 3425 ν (O-H), 2926 and 2845
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ν (C-H), 1741 ν (C=O methyl ester), 1722-1704 ν (C=O fatty acid ester), 1629 νas (COO-), 1439 νs (COO-), 1207 and 1133 ν (C-O); pectin-oleate (2): FT-IR (cm-1): 3424 ν (O-H), 2925 and 2855 ν (C-H), 1747 ν (C=O ester), 1721-1704 ν (C=O fatty acid ester), 1639 νas (COO-), 1443 νs (COO-),
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1207 and 1142 ν (C-O); and pectin-palmitate (3): FT-IR (cm-1): 3445 ν (O-H), 2923 and 2854 ν (CH), 1749 ν (C=O ester), 1723-1705 ν (C=O fatty acid ester), 1635 νas (COO-), 1436 νs (COO-), 1207 and 1133 ν (C-O).
Thermal analysis (TGA) was carried out in air atmosphere with a Mettler TC-10 Thermobalance
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(Novate Milanese, Italy) from room temperature to 1000 °C at a heating rate of 10 °C/min on 10 mg samples in duplicate.
2.4 Antimicrobial assessment 2.4.1 Antimicrobial test The strains used for the antimicrobial assays were the following: the Gram-positive bacterium S. aureus ATCC 6538 and the Gram-negative bacteria E. coli ATCC11219. To standardize the bacterial cell suspension for antibacterial activity assay, some colonies of each strain grown overnight on MHA plates were resuspended in MHB and incubated at 37°C until visible turbidity. This log-phase inoculum was resuspended in 0.9% sterile saline to reach an appropriate OD600
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value (with a Bio-Rad Microplate Reader - Bio-Rad Laboratories, Hercules, CA, USA) corresponding to a concentration of about 1 x 108 CFU/mL. This standardized inoculum was diluted 1:10 in MHB and the inoculum size was confirmed by colony counting.
2.4.2 Antimicrobial Activity Assay Susceptibility testing was performed using the broth microdilution method outlined by the Clinical
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and Laboratory Standards Institute using sterile 96-well microtiter plates (Falcon, NJ, USA). For the quantitative assay of the antimicrobial activity of our compounds by the microdilution method in
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liquid medium distributed in 96-well plates, binary serial dilutions of the tested compounds solutions were performed.
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Each well was then inoculated with 5 µl of the standardized bacterial inoculum, corresponding to a final test concentration of about 5 x 105 CFU/mL. Antimicrobial activities were expressed as the
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percentage value of colony (formation) reduction observed after 24 h of incubation at 37° C.
2.4.3 Eukaryotic cells cytoxicity
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Vero cells were exposed to increasing concentrations of compounds, and the number of viable cells was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
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assay that is based on the reduction of the yellowish MTT to the insoluble and dark blue formazan by viable and metabolically active cells. Vero cells were subcultured in 96-well plates at a seeding density of 2 × 104 cells/well and treated with compounds for 3, 10 and 24 hours. The medium was
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then gently aspirated, MTT solution (5 mg/mL) was added to each well, and cells were incubated for a further 3 hours at 37°C. The medium with MTT solution was removed, and the formazan crystals were dissolved with dimethyl sulfoxide. The absorption values were measured at 570 nm using a Bio-Rad Microplate Reader. The viability of Vero cells in each well was presented as a
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percentage of control cells.
2.5 Coating preparation and SEM analysis The polyethylene (PE) film, 80µm thick and 5cm diameter, was used as polymeric substrate for the coating. The coating solutions were prepared by dissolving 15mg of pectin sample (pectin-palmitate, pectin-oleate, pectin-linoleate) in 20 ml of water, the mixture was heated at 150°C for 1hour. Both sides of PE film were coated by evaporation of the of water from pectin, pectin-palmitic, pectinoleic and pectin-linoleic solution, giving a layer of coating of 0.7-1 µm. PE film was used as control and the coatings resulted mildly adhesive. The morphology of the films was analyzed by scanning electronic microscopy (SEM).
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Pieces of film (10 × 10 mm2) from each samples were cut and fixed in a little support (stub). All samples were sputter coated with gold (Agar Automatic Sputter Coater Mod. B7341, Stansted, UK) at 30 mA for 180 s and micrographs were collected by a FEI Quanta 200 FEG scanning electronic microscope (Eindhoven, The Nederlands).
2.6 UV irradiation test
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Accelerate Weathering tests were carried out using QUV/Spray accelerated weathering tester supplied by Q Panel lab products-USA, at an irradiance of 0.78 W/ m2 at 340 nm and at
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temperature of 45 °C. This is considered a good match with noon summer sunlight. The QUV/Spray tester uses fluorescent UVA-340 lamps that gives a good simulation of sunlight in the critical short
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wavelength region from 365 nm down to the solar cut-off of 295 nm. The UVA-340 lamps are more stable than other types of UV lamps and provide more reproducible test results by the solar eye
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irradiance light control system which continuously monitors and compensate the irradiance level from the lamps. Exposure times were of 1hour, 2 hours, 4 hours, 8 hours, 24 hours, 50 hours, 100
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hours and 200 hours.
3. Results and discussion
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The synthesis of the pectin derivatives was performed by acylation of polysaccharide alcoholic functions with several fatty acids anhydrides. The reaction was performed by mechanical milling of the polysaccharide with the appropriate fatty acid anhydride, in presence of catalytic amount of
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K2CO3 and few drops of ethanol. The obtained mixture was irradiated with microwaves for two cycles of 3 min each (Scheme 1) [10-12].
All unreacted fatty acids were washed away with ethyl acetate and the solid was titrated with 0.5 N HCl until a neutral pH was reached. The resultant salt KCl was removed by dialysis in Milli-Q
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water [10].
The final products were fully characterized by FT-IR spectroscopy. In figure 1a the FT-IR spectra of pectin-oleate, pectin palmitate and pectin-linoleate and, for comparison purpose, a spectrum of the native pectin are reported. The chemical modifications of the polysaccharide produced the main changes of the infrared spectrum: a decrease of the O–H stretching band (in the 3500–3200 cm−1 range) is related to the decrease of the hydroxyl pectin groups upon reaction with the fatty acid anhydrides; an increase in the C–H stretching region (2925–2934 cm−1 for pectin-linoleate; 2925-2930 cm−1 for pectin-oleate; 2919–2926 cm−1 for pectin-palmitate) and the appearance of well-defined band (2845-2854 cm−1 for pectin-linoleate; 2849–2855 for pectin-oleate; 2847–2855 cm−1 for pectin-palmitate) was also
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observed. The region featuring the carboxylic groups (1750–1350 cm−1) is the more interesting [1317]. For each sample the appearance of a new band in the C=O ester stretching region, which overlaps with the pectin methyl ester band, accounted for the esterification of the fatty acid employed (17221704 cm−1 for pectin-linoleate; 1721-1704 cm−1 for pectin-oleate; 1723-1705 cm−1 for pectinpalmitate) (Figure 1b) [12].
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By evaluating the ratio between the pectin methyl ester band and the fatty acid ester band, the samples were judged characterized by a similar degree of substitution [10-12].
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Concerning with the thermogravimetric characterization, the samples, analyzed for their weight loss, showed a degradation path very similar to the pristine pectin, thus proving that the pristine
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structure was preserved after the chemical modifications (Figure 2) [11-12].
The antimicrobial activity of the obtained pectin derivatives was analyzed by the broth
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microdilution method against the Gram-positive bacterium (S. aureus) and the Gram-negative bacteria (E. coli) [18-20]. The results are shown in figure 3 and 4.
Our results have shown that, among the tested fatty acids, oleate and linoleate acids exihibited the
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greatest inhibitory effect. In particular, these compounds were able to inhibit the growth of the selected microorganisms in inhibition zone values between 50 and 70%. However we noticed a
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better antimicrobial activity against S. aureus.
To confirm that these compounds did not exert toxic effects on eukaryotic cells, monolayers of Vero cells were exposed to different amounts (10, 20, and 50 μl) of each compound for 3, 10 and 24
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hours, and cell viability was quantified by the MTT assay [21]. No statistical difference was observed between the viability of control (untreated) cells and that of cells exposed to the compounds up to amounts twofold higher than the once used in antimicrobial testing. On the basis of these results, in order to explore the applicability of the developed materials as
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active packaging system, coatings of the pectin derivatives on polyethylene films were prepared. Indeed, PE is widely employed for packaging materials, due to its low cost, versatile properties, and ease with which it can be manufactured. In particular, PE films were coated on both sides by evaporation of the water from pectin, pectin-palmitate, pectin-oleate and pectin-linoleate solution, giving a layer of coating of 0.7-1 µm [22-24]. The obtained coating surfaces were examined by scanning electron microscopy (SEM). As an example, in figure 5 the images of the pristine PE film and the PE coated with pectin-palmitate are reported. As shown, the PE surface appears very smooth and structureless, whereas the pectin-palmitate coating shows the morphology of the modified pectin, very homogeneously distributed and
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characterized by few defects. This result confirmed that the coating is well distributed on the polyolefin surface. Since active packaging systems have to be endowed of both an oxygen scavenging function as well as an antimicrobial activity, we also evaluated the oxygen barrier properties of the PE films coated with pectin-oleate and pectin-linoleate. In fact, the double bonds, present in both oleic and linoleic acids, are expected to capture the oxygen molecules, reducing their number inside the PE film [25-
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28].
Therefore we conducted an accelerated exposition of the coated films to UV radiation, in the
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presence of air, and monitored the FTIR band at 1640 cm-1, relative to the double bonds. This band was normalized with the band at 2020 cm-1 for eliminating thickness differences. In Figure 6 we
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show the ratio R=A1640/A2020 as a function of the irradiation time in hours for the PE film coated with pectin-oleate.
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We observe that the R index, relative to the presence of double bonds, begins to decrease after 20 hours of exposition and progressively is reduced up to the highest investigated time, that is 200 hours. The continuous decrease of the double bond indirectly indicates that the presence of this
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functionality is able to reduce the oxygen level into polyethylene film.
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4. Conclusions
Our studies assessed that the pectin derivatives, prepared by chemical modification of the polysaccharide with natural fatty acids, are promising and effective antimicrobial agents against
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several foodborne pathogens (Staphylococcus aureus, Escherichia coli). Moreover, the PE films coated with pectin-oleate and pectin-linoleate samples were proven to capture the oxygen, thus reducing its permeability through the polymeric support (polyethylene). These results suggest that the coating of the developed pectin derivatives can find application in the active food packaging
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field. Further studies are currently in progress in order to characterize the bactericidal ability of pectin derivatives coated on polyethylene films.
Acknowledgment The Authors wish to highly acknowledge the contribution of Dr. C. Naddeo for the accelerated exposition of the coated films to UV radiation.
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[3] J. Dutta, S. Tripathi, P. K. Dutta, Food Science and Technology International 18 (2012) 3-34. [4] J. J. Kabara, D. M. Swieczkowski, A. J. Conley, J. P. Truant, Antimicrobial Agents and Chemotherapy 2 (1972) 23-28. [5] H. Zhang, L. Zhang, L.-J. Peng, X.-W. Dong, D. Wu, V. C.-H. Wu, F.-Q. Feng, Journal of Zhejiang University SCIENCE B (Biomedicine & Biotechnology) 13 (2012) 83-93. [6] L. Timofeeva, N. Kleshcheva, Applied Microbiology and Biotechnology 89 (2011) 475-492.
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[7] K. K. Kuorwel, M. J. Cran, K. Sonneveld, J. Miltz, S. W. Bigger, Journal of Food Science 76 (2011) 90-102.
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[10] L. Monfregola, M. Leone, V. Vittoria, P. Amodeo, S. De Luca, Polymer Chemistry 2 (2011) 800-
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[11] L. Monfregola, V. Bugatti, P. Amodeo, S. De Luca, V. Vittoria, Biomacromolecules 12 (2011) 2311-2318.
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[12] E. Calce, V. Bugatti, V. Vittoria, S. De Luca, Molecules 17 (2012) 12234-12242. [13] A. Synytsya, J. Copikova, P. Matejka, V. Machovic. Carbohydrate Polymers 54 (2003) 97-106. [14] M. P. Filippov, Food Hydrocolloids 6 (1992) 115-142.
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[15] A. A. Kamnev, N. M. Ptitchkina, M. Ristic, O. G. Shkodina, V. V. Ignatov, in Spectroscopy of Biological Molecules, ed. J. C. Merlin, S. Turrell, and J. P. Huvenne, Kluwer, 1995a, Dordrecht, pp. 445-446.
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[16] R. G. Zhbankov, Journal of Molecular Structure 275 (1992) 65-84. [17] A. A. Kamnev, M. Colina, J. Rodriguez, N. M. Ptitchkina, V. V. Ignatov, Food Hydrocolloids 12 (1998) 263-271.
[18] O. Scudiero, S. Galdiero, M. Cantisani, R. Di Noto, M. Vitiello, M. Galdiero, G. Naclerio, J. J.
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Cassiman, C. Pedone, G. Castaldo, F. Salvatore, Antimicrobial Agents and Chemotherapy 54 (2010) 2312-2322.
[19] S. Mabrouk, K. B. Salah, A. Elaissi, L. Jlaiel, H. B. Jannet, M. Aouni, F. Harzallah-Skhiri, Chemistry & Biodiversity 10 (2013) 209-223. [20] S. Rashid, M. A. Rather, W. A. Shah, B. A. Bhat, Food Chemistry 138 (2013) 693-700. [21] T. Mosmann, Journal of Immunological Methods 65 (1983) 55-63. [22] I. Arvanitoyannis, C. G. Biliaderis, H. Ogawa, N. Kawasaki, Carbohydrate Polymers 36 (1998) 89104. [23] P. Appendini, J. H. Hotchkiss, Innovative Food Science and Emerging Technologies 3 (2002) 113126. [24] D. Raheem, Emirates Journal of Food and Agriculture 25 (2012) 177-188.
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[25] Y. Maeda, D. R. Paul, Journal of Polymer Science Part B 25 (1987) 1005-1016. [26] M. Salame, The use of barrier polymers in food and beverage packaging, in K. M. Finlayson (Ed.), Plastic film technology, Technomic, vol. 1, 1989, pp. 132-145. [27] G. L. Robertson, Food Packaging: Principles and Practice, Marcel Dekker, 1993, pp. 79-110. [28] V. Bugatti, S. Livi, S. Hayrapetyan, Y. Wang, L. Estevez, V. Vittoria, E. P. Giannelis, Journal of
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Colloid and Interface Science 396 (2013) 47-52.
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Highlights (for review)
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Pectin samples derivatized with fatty acids are promising antimicrobial agents. Pectin functionalized with fatty acids as oxygen scavenger Coating of the pectin derivatives as active food packaging system.
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Figure(s)
COOCH3 O
O
O HO
HO
O
R
COOH O
O O
R
R: (1) O
HO
HO
(2) (3)
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K2CO3 0.1 eq, CH3CH2OH, MW (900 W), 6 min
COOCH3
O
HO
O
COOH O O
O R
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O
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HO
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O
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Scheme 1
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Figure(s)
a)
b)
140
%T
120 100
EP563-1
120
EP563-1
100
4000
80 60
80
1800 40 60
3000
40
2000
1000
Wavenumber [cm]
20
-1
0 4000
1400
1200
Wavenumber [cm]
1000
3500
3000
-1
2500
2000
1500
1000
500
3500
3000
2500
2000
1500
1000
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0 4000
1600
A
20
500
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A
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Figure 1
Pectin Pec-Linoleate Pec-Oleate Pec-Palmitate
140
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%T
Pectin Pec-Linoleate Pec-Oleate Pec-Palmitate
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Figure(s)
110
pectin-linoleate pectin-oleate pectin pectin-palmitate
100 90 80
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60 50 40
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Weight (%)
70
30
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20 10
-10 100
200
300
400
500
600
700
800
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T(°C)
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Figure 2
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Figure(s)
Bacterial growth inibition Staphylococcus Aureus 80% 60%
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40% 20%
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Figure 3
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Figure(s)
Bacterial growth inibition Escherichia Coli 80% 60%
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40% 20%
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0%
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Figure 4
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Figure(s)
a
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b
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Figure 5
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Figure(s)
R1640/2020
3.5
3.0
2.5
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2.0
1.5 50
100
150
200
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0
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UV irradiation time (hours)
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Figure 6
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Figure(s)
Figure captions: Fig. 1 (a) Transmittance FT-IR spectra of pectin derivatives; (b) Expansion of a region of the transmittance FT-IR spectra Fig.2 The loss of weight of the pristine and modified pectin samples as a function of temperature
Fig.4 Inhibition percentage of E. coli growth
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Fig.5 SEM micrographs of PE (a) and PE coated with Pec-Palmitate (b)
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Fig.3 Inhibition percentage of S. aureus growth
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Fig.6 R index in function of irradiation time for PE coated with Pec-oleate
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Scheme 1. Synthesis of pectin samples functionalized with fatty acids
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S0141-8130(14)00238-4 http://dx.doi.org/doi:10.1016/j.ijbiomac.2014.04.011 BIOMAC 4278
To appear in:
International Journal of Biological Macromolecules
Received date: Revised date: Accepted date:
17-12-2013 5-4-2014 7-4-2014
Please cite this article as: E. Calce, E. Mignogna, V. Bugatti, M. Galdiero, V. Vittoriac, S. De Luca, Pectin functionalized with natural fatty acids as antimicrobial agent, International Journal of Biological Macromolecules (2014), http://dx.doi.org/10.1016/j.ijbiomac.2014.04.011 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Graphical abstract
PecPecLinoleate Oleate
80%
PecPalmitate
Pectin
40%
Antimicrobial activity Escherichia Coli
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Staphilococcus Aureus
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0%
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*Manuscript
Pectin functionalized with natural fatty acids as antimicrobial agent Enrica Calce,a Eleonora Mignogna,b Valeria Bugatti,c Massimiliano Galdiero,b Vittoria Vittoriac and Stefania De Luca*a Institute of Biostructures and Bioimaging, National Research Council, 80134 Naples, Italy.
b
Department of Experimental Medicine, II University of Naples, Via De Crecchio 7, 80138, Naples, Italy
c
Department of Industrial Engineering, University of Salerno, 84084 Fisciano (SA), Italy
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a
Corresponding author. Tel: +39 0812534514; fax: +39 0812536642; e-mail: [email protected]
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Abstract
Several pectin derivatives were prepared by chemical modifications of the polysaccharide with natural fatty
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acids. The obtained biodegradable pectin-based materials, pectin-linoleate, pectin-oleate and pectinpalmitate, were investigated for their antimicrobial activity against several bacterial strains, Staphylococcus
an
aureus and Escherichia coli. Good results were obtained for pectin-oleate and pectin-linoleate, which inhibit the growth of the selected microorganisms by 50-70%. They exert the better antimicrobial activity against S. aureus.
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Subsequently, the pectin-oleate and the pectin-linoleate samples were coated on polyethylene films and were assessed for their capacity to capture the oxygen molecules, reducing its penetration into the polymeric support. These results confirmed a possible application of the new materials in the field of active food
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packaging.
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Keywords: chemical modification, fatty acid ester of pectin, antimicrobial activity, oxygen scavenging function, active packaging systems
1. Introduction
During the past decade, an increasing interest has been devoted to the development of antimicrobial
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systems to be used as biocides in various areas. In particular, recyclability and biodegradability are nowadays considered important issues when new products with antimicrobial properties are introduced for various applications in medical, pharmaceutical, agriculture, and packaging fields [13]. Free fatty acids (FFA) can be considered natural products, since they are usually provided by natural resources, like triglycerides or phospholipids, and for this reason can be employed with the great advantage of low environmental impact. Within their broad spectrum of biological activities, free fatty acids are able to kill or inhibit the growth of several pathogenic bacteria [4,5]. Whilst their antibacterial mode of action is still unclear, fatty acids have as prime target the bacterial cell membrane and the various essential processes that occur within and at the membrane. Other
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processes that may contribute to bacterial growth inhibition or death include cell lysis, inhibition of enzyme activity, impairment of nutrient uptake and the generation of toxic peroxidation and autooxidation product [6]. However, the usage of FFA as antibacterial agents suffer of several limitations, since some FFAs have an unpleasant taste, while others can be unstable and also have a tendency to bind nonspecifically to proteins. An alternative route for exploring the commercial potential of FFA as antimicrobials could be the conjugation of them to specific carriers, like
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biodegradable material, in order to modulate their delivery. In this regard, biodegradable polymers, produced from natural, renewable resources, can represent ideal candidates, since they readily
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decompose thus reducing the negative environment impact [7-9]. We have recently developed a synthetic process to chemically modify the pectin from apple via acylation of its alcoholic functions
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with several natural fatty acids [10-12]. The aim was to generate new materials by using renewable resources and limiting the number of chemicals and reaction steps. Indeed, natural polymers suffer
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of low water resistance and poor mechanical properties (tensile strength and elongation to break), and these drawbacks are limiting factors for their use as manufactured materials. The chemical modification of pectin with fatty acids as a matter of fact improved water resistance and barrier
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properties of the polysaccharide, thus generating new bio-based material for novel applications. Due to the introduction of fatty acids, these pectin derivatives can be studied for their antimicrobial
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properties, and eventually considered as coatings to be used for the food packaging. With these ideas in our mind, we decided to prepare several pectin samples functionalized with different fatty acids (palmitic, oleic and linoleic acid) in order to investigate their antimicrobial
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activity against two foodborne phatogens, Staphylococcus aureus (Gram-positive) and Escherichia coli (Gram-negative) [8].
2. Experimental
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2.1 Materials
The apple peel Pectin was purchased from Fluka. It is a powder sample with high molecular weight (30 000-100 000 g/mol) and a high degree of esterification (70-75%) on a dry basis. The fatty acids and all solvents were purchased from Sigma-Aldrich.
2.2 Synthesis of pectin derivatives Synthesis of fatty acid anhydrides was performed as follows. The appropriate fatty acid (10 mmol) was dissolved in dichloromethane (2 mL), the solution was cooled in an ice-water bath and stirred vigorously under argon atmosphere. The dicyclohexylcarbodiimide (5 mmol), previously dissolved in the minimum volume of dichloromethane, was added and stirring was continued at ice bath
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temperature for 2 h. The white solid N,N’-dicyclohexylurea was removed by filtration and the solvent was evaporated in vacuo to give the final anhydride [10]. By using an agate mortar, 30 mg of pectin were manually milled with 30 mg of the appropriate fatty acid anhydride in the presence of K2CO3 (0.1 equiv) and few drops of ethanol to obtain the different pectin-derived materials. Reactions were carried out in a domestic microwave oven and irradiated with microwaves (900 W) for two cycles of 3 minutes each. After cooling at room temperature, the
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final crude product was washed with ethyl acetate. The obtained solid was dissolved in water, and 0.5 N HCl was added to the final solution until a neutral pH was reached. This solution was then
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dialyzed (membrane cut off 6000-8000) for 1 day in Milli-Q water and finally lyophilized to give
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the desired product.
2.3 Characterization
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All modified pectin samples were analyzed by FT-IR spectroscopy. The FT-IR spectra were recorded on a Jasco spectrometer. Samples were ground into a fine powder using an agate mortar before being compressed into KBr discs. The characteristic peaks of IR transmission spectra were
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recorded at a resolution of 4 cm-1 over a wavenumber region of 400-4000 cm-1. The bands relevant for the structural organization are: pectin-linoleate (1): FT-IR (cm-1): 3425 ν (O-H), 2926 and 2845
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ν (C-H), 1741 ν (C=O methyl ester), 1722-1704 ν (C=O fatty acid ester), 1629 νas (COO-), 1439 νs (COO-), 1207 and 1133 ν (C-O); pectin-oleate (2): FT-IR (cm-1): 3424 ν (O-H), 2925 and 2855 ν (C-H), 1747 ν (C=O ester), 1721-1704 ν (C=O fatty acid ester), 1639 νas (COO-), 1443 νs (COO-),
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1207 and 1142 ν (C-O); and pectin-palmitate (3): FT-IR (cm-1): 3445 ν (O-H), 2923 and 2854 ν (CH), 1749 ν (C=O ester), 1723-1705 ν (C=O fatty acid ester), 1635 νas (COO-), 1436 νs (COO-), 1207 and 1133 ν (C-O).
Thermal analysis (TGA) was carried out in air atmosphere with a Mettler TC-10 Thermobalance
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(Novate Milanese, Italy) from room temperature to 1000 °C at a heating rate of 10 °C/min on 10 mg samples in duplicate.
2.4 Antimicrobial assessment 2.4.1 Antimicrobial test The strains used for the antimicrobial assays were the following: the Gram-positive bacterium S. aureus ATCC 6538 and the Gram-negative bacteria E. coli ATCC11219. To standardize the bacterial cell suspension for antibacterial activity assay, some colonies of each strain grown overnight on MHA plates were resuspended in MHB and incubated at 37°C until visible turbidity. This log-phase inoculum was resuspended in 0.9% sterile saline to reach an appropriate OD600
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value (with a Bio-Rad Microplate Reader - Bio-Rad Laboratories, Hercules, CA, USA) corresponding to a concentration of about 1 x 108 CFU/mL. This standardized inoculum was diluted 1:10 in MHB and the inoculum size was confirmed by colony counting.
2.4.2 Antimicrobial Activity Assay Susceptibility testing was performed using the broth microdilution method outlined by the Clinical
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and Laboratory Standards Institute using sterile 96-well microtiter plates (Falcon, NJ, USA). For the quantitative assay of the antimicrobial activity of our compounds by the microdilution method in
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liquid medium distributed in 96-well plates, binary serial dilutions of the tested compounds solutions were performed.
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Each well was then inoculated with 5 µl of the standardized bacterial inoculum, corresponding to a final test concentration of about 5 x 105 CFU/mL. Antimicrobial activities were expressed as the
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percentage value of colony (formation) reduction observed after 24 h of incubation at 37° C.
2.4.3 Eukaryotic cells cytoxicity
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Vero cells were exposed to increasing concentrations of compounds, and the number of viable cells was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
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assay that is based on the reduction of the yellowish MTT to the insoluble and dark blue formazan by viable and metabolically active cells. Vero cells were subcultured in 96-well plates at a seeding density of 2 × 104 cells/well and treated with compounds for 3, 10 and 24 hours. The medium was
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then gently aspirated, MTT solution (5 mg/mL) was added to each well, and cells were incubated for a further 3 hours at 37°C. The medium with MTT solution was removed, and the formazan crystals were dissolved with dimethyl sulfoxide. The absorption values were measured at 570 nm using a Bio-Rad Microplate Reader. The viability of Vero cells in each well was presented as a
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percentage of control cells.
2.5 Coating preparation and SEM analysis The polyethylene (PE) film, 80µm thick and 5cm diameter, was used as polymeric substrate for the coating. The coating solutions were prepared by dissolving 15mg of pectin sample (pectin-palmitate, pectin-oleate, pectin-linoleate) in 20 ml of water, the mixture was heated at 150°C for 1hour. Both sides of PE film were coated by evaporation of the of water from pectin, pectin-palmitic, pectinoleic and pectin-linoleic solution, giving a layer of coating of 0.7-1 µm. PE film was used as control and the coatings resulted mildly adhesive. The morphology of the films was analyzed by scanning electronic microscopy (SEM).
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Pieces of film (10 × 10 mm2) from each samples were cut and fixed in a little support (stub). All samples were sputter coated with gold (Agar Automatic Sputter Coater Mod. B7341, Stansted, UK) at 30 mA for 180 s and micrographs were collected by a FEI Quanta 200 FEG scanning electronic microscope (Eindhoven, The Nederlands).
2.6 UV irradiation test
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Accelerate Weathering tests were carried out using QUV/Spray accelerated weathering tester supplied by Q Panel lab products-USA, at an irradiance of 0.78 W/ m2 at 340 nm and at
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temperature of 45 °C. This is considered a good match with noon summer sunlight. The QUV/Spray tester uses fluorescent UVA-340 lamps that gives a good simulation of sunlight in the critical short
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wavelength region from 365 nm down to the solar cut-off of 295 nm. The UVA-340 lamps are more stable than other types of UV lamps and provide more reproducible test results by the solar eye
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irradiance light control system which continuously monitors and compensate the irradiance level from the lamps. Exposure times were of 1hour, 2 hours, 4 hours, 8 hours, 24 hours, 50 hours, 100
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hours and 200 hours.
3. Results and discussion
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The synthesis of the pectin derivatives was performed by acylation of polysaccharide alcoholic functions with several fatty acids anhydrides. The reaction was performed by mechanical milling of the polysaccharide with the appropriate fatty acid anhydride, in presence of catalytic amount of
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K2CO3 and few drops of ethanol. The obtained mixture was irradiated with microwaves for two cycles of 3 min each (Scheme 1) [10-12].
All unreacted fatty acids were washed away with ethyl acetate and the solid was titrated with 0.5 N HCl until a neutral pH was reached. The resultant salt KCl was removed by dialysis in Milli-Q
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water [10].
The final products were fully characterized by FT-IR spectroscopy. In figure 1a the FT-IR spectra of pectin-oleate, pectin palmitate and pectin-linoleate and, for comparison purpose, a spectrum of the native pectin are reported. The chemical modifications of the polysaccharide produced the main changes of the infrared spectrum: a decrease of the O–H stretching band (in the 3500–3200 cm−1 range) is related to the decrease of the hydroxyl pectin groups upon reaction with the fatty acid anhydrides; an increase in the C–H stretching region (2925–2934 cm−1 for pectin-linoleate; 2925-2930 cm−1 for pectin-oleate; 2919–2926 cm−1 for pectin-palmitate) and the appearance of well-defined band (2845-2854 cm−1 for pectin-linoleate; 2849–2855 for pectin-oleate; 2847–2855 cm−1 for pectin-palmitate) was also
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observed. The region featuring the carboxylic groups (1750–1350 cm−1) is the more interesting [1317]. For each sample the appearance of a new band in the C=O ester stretching region, which overlaps with the pectin methyl ester band, accounted for the esterification of the fatty acid employed (17221704 cm−1 for pectin-linoleate; 1721-1704 cm−1 for pectin-oleate; 1723-1705 cm−1 for pectinpalmitate) (Figure 1b) [12].
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By evaluating the ratio between the pectin methyl ester band and the fatty acid ester band, the samples were judged characterized by a similar degree of substitution [10-12].
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Concerning with the thermogravimetric characterization, the samples, analyzed for their weight loss, showed a degradation path very similar to the pristine pectin, thus proving that the pristine
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structure was preserved after the chemical modifications (Figure 2) [11-12].
The antimicrobial activity of the obtained pectin derivatives was analyzed by the broth
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microdilution method against the Gram-positive bacterium (S. aureus) and the Gram-negative bacteria (E. coli) [18-20]. The results are shown in figure 3 and 4.
Our results have shown that, among the tested fatty acids, oleate and linoleate acids exihibited the
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greatest inhibitory effect. In particular, these compounds were able to inhibit the growth of the selected microorganisms in inhibition zone values between 50 and 70%. However we noticed a
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better antimicrobial activity against S. aureus.
To confirm that these compounds did not exert toxic effects on eukaryotic cells, monolayers of Vero cells were exposed to different amounts (10, 20, and 50 μl) of each compound for 3, 10 and 24
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hours, and cell viability was quantified by the MTT assay [21]. No statistical difference was observed between the viability of control (untreated) cells and that of cells exposed to the compounds up to amounts twofold higher than the once used in antimicrobial testing. On the basis of these results, in order to explore the applicability of the developed materials as
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active packaging system, coatings of the pectin derivatives on polyethylene films were prepared. Indeed, PE is widely employed for packaging materials, due to its low cost, versatile properties, and ease with which it can be manufactured. In particular, PE films were coated on both sides by evaporation of the water from pectin, pectin-palmitate, pectin-oleate and pectin-linoleate solution, giving a layer of coating of 0.7-1 µm [22-24]. The obtained coating surfaces were examined by scanning electron microscopy (SEM). As an example, in figure 5 the images of the pristine PE film and the PE coated with pectin-palmitate are reported. As shown, the PE surface appears very smooth and structureless, whereas the pectin-palmitate coating shows the morphology of the modified pectin, very homogeneously distributed and
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characterized by few defects. This result confirmed that the coating is well distributed on the polyolefin surface. Since active packaging systems have to be endowed of both an oxygen scavenging function as well as an antimicrobial activity, we also evaluated the oxygen barrier properties of the PE films coated with pectin-oleate and pectin-linoleate. In fact, the double bonds, present in both oleic and linoleic acids, are expected to capture the oxygen molecules, reducing their number inside the PE film [25-
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28].
Therefore we conducted an accelerated exposition of the coated films to UV radiation, in the
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presence of air, and monitored the FTIR band at 1640 cm-1, relative to the double bonds. This band was normalized with the band at 2020 cm-1 for eliminating thickness differences. In Figure 6 we
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show the ratio R=A1640/A2020 as a function of the irradiation time in hours for the PE film coated with pectin-oleate.
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We observe that the R index, relative to the presence of double bonds, begins to decrease after 20 hours of exposition and progressively is reduced up to the highest investigated time, that is 200 hours. The continuous decrease of the double bond indirectly indicates that the presence of this
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functionality is able to reduce the oxygen level into polyethylene film.
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4. Conclusions
Our studies assessed that the pectin derivatives, prepared by chemical modification of the polysaccharide with natural fatty acids, are promising and effective antimicrobial agents against
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several foodborne pathogens (Staphylococcus aureus, Escherichia coli). Moreover, the PE films coated with pectin-oleate and pectin-linoleate samples were proven to capture the oxygen, thus reducing its permeability through the polymeric support (polyethylene). These results suggest that the coating of the developed pectin derivatives can find application in the active food packaging
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field. Further studies are currently in progress in order to characterize the bactericidal ability of pectin derivatives coated on polyethylene films.
Acknowledgment The Authors wish to highly acknowledge the contribution of Dr. C. Naddeo for the accelerated exposition of the coated films to UV radiation.
References [1] E.-R. Kenawy, S. D. Worley, R. Broughton, Biomacromolecules 8 (2007) 1359-1384. [2] N. Bordenave, S. Grelier, V. Coma, Biomacromolecules 11 (2010) 88-96. Page 8 of 19
[3] J. Dutta, S. Tripathi, P. K. Dutta, Food Science and Technology International 18 (2012) 3-34. [4] J. J. Kabara, D. M. Swieczkowski, A. J. Conley, J. P. Truant, Antimicrobial Agents and Chemotherapy 2 (1972) 23-28. [5] H. Zhang, L. Zhang, L.-J. Peng, X.-W. Dong, D. Wu, V. C.-H. Wu, F.-Q. Feng, Journal of Zhejiang University SCIENCE B (Biomedicine & Biotechnology) 13 (2012) 83-93. [6] L. Timofeeva, N. Kleshcheva, Applied Microbiology and Biotechnology 89 (2011) 475-492.
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[7] K. K. Kuorwel, M. J. Cran, K. Sonneveld, J. Miltz, S. W. Bigger, Journal of Food Science 76 (2011) 90-102.
[8] L. Fan, M. Cao, S. Gao, W. Wang, K. Peng, C. Tan, F. Wen, S. Tao, W. Xie, Carbohydrate Polymers
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[9] G. Geisberger, E. Besic Gyenge, D. Hinger, A. Kach, C. Maake, G. R. Patzke, Biomacromolecules
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[10] L. Monfregola, M. Leone, V. Vittoria, P. Amodeo, S. De Luca, Polymer Chemistry 2 (2011) 800-
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[11] L. Monfregola, V. Bugatti, P. Amodeo, S. De Luca, V. Vittoria, Biomacromolecules 12 (2011) 2311-2318.
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[12] E. Calce, V. Bugatti, V. Vittoria, S. De Luca, Molecules 17 (2012) 12234-12242. [13] A. Synytsya, J. Copikova, P. Matejka, V. Machovic. Carbohydrate Polymers 54 (2003) 97-106. [14] M. P. Filippov, Food Hydrocolloids 6 (1992) 115-142.
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[15] A. A. Kamnev, N. M. Ptitchkina, M. Ristic, O. G. Shkodina, V. V. Ignatov, in Spectroscopy of Biological Molecules, ed. J. C. Merlin, S. Turrell, and J. P. Huvenne, Kluwer, 1995a, Dordrecht, pp. 445-446.
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[16] R. G. Zhbankov, Journal of Molecular Structure 275 (1992) 65-84. [17] A. A. Kamnev, M. Colina, J. Rodriguez, N. M. Ptitchkina, V. V. Ignatov, Food Hydrocolloids 12 (1998) 263-271.
[18] O. Scudiero, S. Galdiero, M. Cantisani, R. Di Noto, M. Vitiello, M. Galdiero, G. Naclerio, J. J.
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Cassiman, C. Pedone, G. Castaldo, F. Salvatore, Antimicrobial Agents and Chemotherapy 54 (2010) 2312-2322.
[19] S. Mabrouk, K. B. Salah, A. Elaissi, L. Jlaiel, H. B. Jannet, M. Aouni, F. Harzallah-Skhiri, Chemistry & Biodiversity 10 (2013) 209-223. [20] S. Rashid, M. A. Rather, W. A. Shah, B. A. Bhat, Food Chemistry 138 (2013) 693-700. [21] T. Mosmann, Journal of Immunological Methods 65 (1983) 55-63. [22] I. Arvanitoyannis, C. G. Biliaderis, H. Ogawa, N. Kawasaki, Carbohydrate Polymers 36 (1998) 89104. [23] P. Appendini, J. H. Hotchkiss, Innovative Food Science and Emerging Technologies 3 (2002) 113126. [24] D. Raheem, Emirates Journal of Food and Agriculture 25 (2012) 177-188.
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[25] Y. Maeda, D. R. Paul, Journal of Polymer Science Part B 25 (1987) 1005-1016. [26] M. Salame, The use of barrier polymers in food and beverage packaging, in K. M. Finlayson (Ed.), Plastic film technology, Technomic, vol. 1, 1989, pp. 132-145. [27] G. L. Robertson, Food Packaging: Principles and Practice, Marcel Dekker, 1993, pp. 79-110. [28] V. Bugatti, S. Livi, S. Hayrapetyan, Y. Wang, L. Estevez, V. Vittoria, E. P. Giannelis, Journal of
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Colloid and Interface Science 396 (2013) 47-52.
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Highlights (for review)
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Pectin samples derivatized with fatty acids are promising antimicrobial agents. Pectin functionalized with fatty acids as oxygen scavenger Coating of the pectin derivatives as active food packaging system.
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Figure(s)
COOCH3 O
O
O HO
HO
O
R
COOH O
O O
R
R: (1) O
HO
HO
(2) (3)
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K2CO3 0.1 eq, CH3CH2OH, MW (900 W), 6 min
COOCH3
O
HO
O
COOH O O
O R
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O
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HO
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O
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Scheme 1
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Figure(s)
a)
b)
140
%T
120 100
EP563-1
120
EP563-1
100
4000
80 60
80
1800 40 60
3000
40
2000
1000
Wavenumber [cm]
20
-1
0 4000
1400
1200
Wavenumber [cm]
1000
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-1
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3500
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0 4000
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A
20
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A
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Figure 1
Pectin Pec-Linoleate Pec-Oleate Pec-Palmitate
140
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%T
Pectin Pec-Linoleate Pec-Oleate Pec-Palmitate
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Figure(s)
110
pectin-linoleate pectin-oleate pectin pectin-palmitate
100 90 80
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60 50 40
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Weight (%)
70
30
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T(°C)
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Figure 2
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Figure(s)
Bacterial growth inibition Staphylococcus Aureus 80% 60%
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40% 20%
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Figure 3
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Figure(s)
Bacterial growth inibition Escherichia Coli 80% 60%
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40% 20%
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Figure 4
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Figure(s)
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Figure 5
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Figure(s)
R1640/2020
3.5
3.0
2.5
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2.0
1.5 50
100
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UV irradiation time (hours)
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Figure 6
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Figure(s)
Figure captions: Fig. 1 (a) Transmittance FT-IR spectra of pectin derivatives; (b) Expansion of a region of the transmittance FT-IR spectra Fig.2 The loss of weight of the pristine and modified pectin samples as a function of temperature
Fig.4 Inhibition percentage of E. coli growth
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Fig.5 SEM micrographs of PE (a) and PE coated with Pec-Palmitate (b)
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Fig.3 Inhibition percentage of S. aureus growth
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Fig.6 R index in function of irradiation time for PE coated with Pec-oleate
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Scheme 1. Synthesis of pectin samples functionalized with fatty acids
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