Antimicrobial and Physicochemical Characterization of Biodegradable, Nitric Oxide-Releasing Nanocellulose-Chitosan Packaging Membranes

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Article
Antimicrobial and Physicochemical Characterization of Biodegradable,
Nitric Oxide-Releasing Nanocellulose-Chitosan Packaging Membranes
Jaya Sundaram, Jitendra Pant, Marcus James Goudie, Sudhagar Mani, and Hitesh Handa
J. Agric. Food Chem., Just Accepted Manuscript DOI: 10.1021/acs.jafc.6b01936 Publication Date (Web): 03 Jun 2016
Downloaded from http://pubs.acs.org on June 8, 2016
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Page 1 of 29 Journal of Agricultural and Food Chemistry
1 Antimicrobial and Physicochemical Characterization of Biodegradable, Nitric Oxide-
2 Releasing Nanocellulose-Chitosan Packaging Membranes
4 Jaya Sundaram*, Jitendra Pant, Marcus J. Goudie, Sudhagar Mani, Hitesh Handa*
5 School of Biological and Biochemical Engineering, College of Engineering, University of
6 Georgia, Athens, GA, USA
8 *Corresponding Authors:
9 Jaya Sundaram
10 College of Engineering
11 University of Georgia
12 597 D W Brooks Dr
13 Athens, GA 30602
14 Telephone: (706) 542-3815
15 E-mail: jayas@engr,uga.edu
16
17 Hitesh Handa
18 College of Engineering
19 University of Georgia
20 220 Riverbend Road
21 Athens, GA 30602
22 Telephone: (706) 542-8109
23 E-mail: hhanda@uga.edu
1
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry Page 2 of 29
24 Abstract
25 Biodegradable composite membranes with antimicrobial properties consisting of nanocellulose
26 fibrils, chitosan, and S-Nitroso-N-acetylpenicillamine (SNAP) were developed and tested for
27 food packaging applications. Nitric oxide donor, SNAP was encapsulated into completely
28 dispersed chitosan in 100 mL, 0.1N acetic acid and was thoroughly mixed with nanocellulose
29 fibrils (CNF) to produce a composite membrane. The fabricated membranes had a uniform
30 dispersion of chitosan and SNAP within the nanocellulose fibrils, which was confirmed through
31 Scanning Electron Microscopy (SEM) micrographs and chemiluminescence nitric oxide
32 analyzer. The membranes prepared without SNAP showed low water vapor permeability than
33 that of the membranes with SNAP. The addition of SNAP resulted in a decrease in the Youngs
34 modulus for both 2-layer and 3-layer membrane configurations. Antimicrobial property
35 evaluation of SNAP incorporated membranes showed an effective zone of inhibition against
36 bacterial strains of Enterococcus faecalis, Staphylococcus aureus, and Listeria monocytogenes
37 and demonstrated its potential applications for food packaging.
38 Keywords: nanocellulose fibrils; nitric oxide; SNAP; chitosan; antimicrobial; biodegradable;
39 packaging membranes; zone of inhibition
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46
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47 Introduction
48 The Centers for Disease Control and Prevention (CDC), estimates that one in every six
49 Americans gets sick from foodborne illness each year, results in 128,000 hospitalizations and
50 3,000 deaths. Foodborne pathogens deteriorate the quality of food, causing increased food
51 wastage. Recent foodborne microbial outbreaks are driving a search for innovative ways to
52 inhibit microbial growth in foods while maintaining quality, freshness, and safety and extend the
53 shelf-life of fresh produces. The Federal government advises and encourages healthy eating
54 habits, which includes consumption of a variety of fresh fruits and vegetables1, 2. As a result, the
55 per capita consumption of eating fresh produce has increased to $12 billion annual sales in the
56 past few years3,4 and the fresh-cut food industry sector becomes the fastest growing segment of
57 food industries. As the fresh-cut produce market continues to grow, such producers face the
58 challenge of an increase in microbial safety for longer shelf life. From 1996 to 2006, 22
59 foodborne illness outbreaks were associated with the consumption of fresh produce. Of these
60 outbreaks, according to Food and Drug Administration (FDA), 18 outbreaks were implicated by
61 fresh-cut produce. Foodborne illness outbreaks also impact the fresh produce trade leading to
62 economic losses4. Food related epidemic can be prevented by means of improved surveillance
63 and detection of contaminations, enhanced epidemiological investigation, safe packaging, and
64 effective methods to identify pathogens5-10.
65 The packaging of fresh fruits and vegetables is one of the most important steps in the long
66 supply chain from the producer to the consumer. Protecting the fresh produce by packing them in
67 antimicrobial (AM) films can extend their shelf life of food11-14. They also effectively control the
68 foodborne pathogens and food-spoiling microorganisms. These packages are typically
69 manufactured by incorporating antimicrobial agents, immobilized or coated on the surface of the
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70 packaging material. Even though the AM packaging films and number of antimicrobial agents
71 have been studied for many years, commercial successes of these packaging materials are very
72 limited due to many constrains in large scale production15. Selection of packaging system and the
73 antimicrobial agents are very critical as they would influence the inherent physicochemical
74 properties of food. Now there is also an increasing demand for green labeling and environmental
75 safety, leading to an increasing number of R & D efforts in the field of biodegradable food
76 packaging materials. Instead of using polymer materials derived from petroleum products,
77 biopolymers derived from renewable sources (starch, cellulose, protein etc.) are more favorable
78 in developing an eco-friendly packaging system for food. When the antimicrobial agents are
79 combined with biodegradable packaging materials, it features the merits of the packaging system
80 in terms of food safety, shelf-life, and environmental friendliness.
81 Cellulose is one of the most abundantly available biopolymers. The cellulose pulp derived from
82 plants and trees are either mechanically or chemically fibrillated into nanocellulose fibrils (CNF)
83 having 5-20 nm diameter. These nanocellulose fibrils highly influence the properties and
84 functionality of the final products16-19. George et al made food packaging membrane using
85 nanocellulose derived from bacterial cellulose and demonstrated its relevance as a packaging
86 material for the food industry20. The prepared membrane possessed minimal oxygen
87 permeability, good mechanical stability, and controlled water permeability, which are critical for
88 food packaging materials to maintain the quality of packed food. Reinforcing nanocellulose with
89 other long chain polymers such as chitosan to develop biodegradable nanocomposite food
90 packaging materials can further improve the quality of the packaging material as well as long
91 storage life21. Chitosan is a linear polysaccharide, deacetylated derivative of chitin, which is the
92 second most abundant polysaccharide found in nature after cellulose. Chitosan is nontoxic,
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93 biodegradable, and biocompatible with antimicrobial characteristics as well and it could be used
94 as a packaging material for the quality preservation of food22, 23, 24.
95 Antimicrobial films with controlled release of antimicrobial agents are more advantageous
96 than dipping or spraying the food with antimicrobial agents containing edible polymers25-27. In
97 these types of coatings, the antimicrobial activity is lost due to its inactivation by the food
98 components leading to concentrations dropping below active levels28. There are several
99 antimicrobial agents available to incorporate into packaging films. However, each one has its
100 own disadvantages apart from their noted advantages. Sodium nitrite salt has been used for
101 centuries in meat curing. Nitric oxide (NO) released from this salt terminates free radicals
102 present in lipid oxidation and provide the typical property for the cured meat29. There are many
103 NO-donating compounds such as nitrates and nitrites that have been used for several years in
104 curing and preserving meats, fish, and certain cheeses30, 31. Nitric oxide inhibits the growth of
105 wide varieties of bacteria (both gram positive and gram negative), viruses, fungi, and yeast32-34.
106 The biggest advantage of NO as an antimicrobial agent is its antimicrobial activity against
107 antibiotic-resistant strains35-37. However, incorporating NO donor compounds such as S-
108 nitrosothiols in the packaging materials for food has not been yet studied. In this study, we
109 investigated the effects of incorporating the NO donor, S-Nitroso-N-acetylpenicillamine (SNAP)
110 into biodegradable nanocellulose-chitosan composite membranes for increased antimicrobial
111 activity for potential food packaging application.
112 Materials and Methods
113 Chitosan (85% deacetylated) derived from shrimp shell was purchased from Thermo- Fischer
114 (Waltham, MA USA), glacial acetic acid, glycerol, N-acetylpenicillamine, sulfuric acid,
115 hydrochloric acid, sodium nitrite, potassium chloride, sodium chloride, potassium phosphate
5
116 monobasic and sodium phosphate dibasic were purchased from Sigma-Aldrich (St. Louis, MO,
117 USA). Nanocellulose fibrils were bought from the University of Maine, MI. Luria broth (LB)-
118 Lennox and Luria Agar (LA) - Miller were obtained from Fisher Bioreagents (Fair Lawn, NJ).
119 Synthesis of S-Nitroso-N-acetyl-D-penicillamine (SNAP)
120 S-Nitroso- acetyl penicillamine was synthesized using a modified version of a previously
121 reported method by Clough and Thrush (1967). Equimolar ratios of N-acetylpenicillamine (NAP)
122 and sodium nitrite were added to a reaction vessel, which had 1:1 mixture of water and methanol
123 containing 2 M HCl and 2 M H2SO4, and stirred for 30 minutes. The reaction vessel was cooled
124 in an ice bath to precipitate the SNAP crystals. After precipitation of SNAP crystals, they were
125 collected by filtration and washed with water to remove unreacted salts, and air dried. The entire
126 process of SNAP synthesis was protected from light. The purity level of synthesized SNAP was
127 tested using the Sievers Chemiluminescence Nitric Oxide Analyzer and recorded greater than
128 95%.
129 Fabrication of composite membrane
130 Biodegradable antimicrobial packaging membranes were prepared in 2-layer and 3-layer
131 configurations. The 2-layer membranes contained one layer with 2 wt% SNAP (antimicrobial
132 agent) in nanocellulose-chitosan with a top layer of nanocellulose-chitosan mix without SNAP.
133 Similarly, the 3-layer membranes contained the middle layer with 2 wt% SNAP in
134 nanocellulose-chitosan with a top and bottom layers of nanocellulose-chitosan mix without
135 SNAP. The control membranes of 2-layer and 3-layer configurations were prepared separately
136 using nanocellulose-chitosan mix, without SNAP antimicrobial agent. About 2 wt % of chitosan
137 was completely dispersed in 100 mL of 0.1N acetic acid at room temperature (212oC) using
138 magnetic stirrer plate. In another beaker 2 wt% of nanocellulose fibrils (CNF) was thoroughly
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139 dispersed in 100 mL of distilled (DI) water using magnetic stirrer plate. Completely dispersed
140 CNF and chitosan were combined together and mixed homogeneously; 3 mL of 80% glycerol
141 was added as a plasticizing agent while mixing them together. The resulting mixture was
142 degassed and poured into a clean glass petri dish of 14 cm diameter and air dried. While the
143 drying was incomplete, another set of membrane solution was made as mentioned above and
144 poured on the partly dried membrane as a second layer after 24 hours. On average it took 48
145 hours to dry for 2-layer control membrane. Similarly, 3-layer control membrane was also made
146 as 2-layer control membrane with additional layer of CNF-chitosan mix and additional drying
147 time of 24 h; in total approximately 72 h to complete the processing of 3-layer configuration.
148 To prepare NO-releasing antimicrobial membranes, 2 wt % of chitosan was completely
149 dispersed in 100 mL of 0.1N acetic acid as said above. 2 wt% SNAP was dissolved in 80%
150 ethanol and 3 mL of the resulting solution was added into the completely dispersed chitosan and
151 the mixing process was continued to obtain complete encapsulation of SNAP. In another beaker
152 2 wt% of nanocellulose fibrils (CNF) was thoroughly dispersed in 100 mL of distilled (DI) water
153 as mentioned in control membrane fabrication. Completely dispersed CNF and chitosan with
154 SNAP were then homogeneously mixed together; while mixing them, 3 mL of 80% glycerol was
155 added as a plasticizing agent. This mix was degassed before casting the membrane. The 2-layer
156 antimicrobial membranes were prepared by casting membrane without SNAP first and after 24
157 hours of drying, SNAP mixed chitosan and CNF homogeneous mix was poured on top of the
158 partially dried membrane of without SNAP and air dried completely. For 3-layer membranes, the
159 middle layer was made with SNAP and top and bottom were made without SNAP in an order
160 such as CNF-chitosan mix without SNAP first, CNF-chitosan mix with SNAP second and again
161 CNF-chitosan mix without SNAP third.
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162 Physicochemical characterization
163 Water permeability
164 To determine water vapor permeability of the composite membranes the procedure followed
165 by Jaya and Das38 was used. Approximately 5 g of dehydrated silica gel was filled separately in 6
166 glass vials of uniform volume. The lid was replaced by one of the 6 membranes (2-layer with
167 SNAP, 3-layer with SNAP, 2-layer control, 3-layer control, control chitosan and control CNF).
168 All the vials were weighed and then placed in an environment maintained at 75% relative
169 humidity (RH) and 222o C established with saturated sodium chloride solution in a desiccator39.
170 The weight of each vial containing the silica gel was recorded at 24 h intervals for 7 days, and
171 the mean weight gained by the silica gel was calculated for each day. The water vapor
172 permeability, K (kg. m/ m2.day.Pa), of the composite membrane was calculated using the
173 following equation.
/
174 =

175 where / is the slope of curve plotted between the time (day) and cumulative moisture
176 gain; w (kg) is the weight of the silica gel packed in the vials; t is the thickness of the film (m);
177 (m2) is the surface area of the composite membranes; (Pa) is the saturation vapor pressure
178 of water at 22oC; the temperature of the environment chosen for the experiment and the relative
179 humidity (75%).
180 Tensile strength
181 Mechanical attributes of the composite membranes were characterized in terms of tensile
182 force using an Instron material testing machine (Instron model 5545, USA) with 1kN load cell.
183 Test specimens were cut according to the IPC-TM-650 standards with a 6:1 length to width ratio.
184 Gauge length and thickness values were recorded individually for each sample, and carefully
8
185 aligned to ensure minimal torsional forces were applied, and held by pneumatic jaws with 1x1
186 against 1x3 rubber faces. Specimens were tested at a constant extension rate of the cross head
187 speed of 1 mm/s. The tests were done at 23 2C and 50 5% RH. Data points of force,
188 distance and time were collected and analyzed for stress and strain relationships. Youngs moduli
189 of the composite membranes were derived from the stress and strain relationships. Tensile
190 strength and Youngs modulus were then calculated for each sample and averaged for each
191 material. All materials were tested in triplicate.
192 Morphology study using scanning electron microscopy
193 Film surface morphology and microstructure were examined using scanning electron
194 microscopy (SEM) (FEI Inspect F FEG-SEM). Dried film samples were mounted on a metal stub
195 with double-sided carbon tape and sputter coated with 10 nm gold-palladium using a Leica EM
196 ACE200 sputter coater. Images were taken at accelerating voltage 20 kV and a magnification of
197 2000X.
198 NO release measurements
199 Nitric oxide release from the chitosan-nanocellulose composites were measured using a
200 Sievers Chemiluminescence Nitric Oxide Analyzer (NOA) model 280i (Boulder, CO). The NOA
201 has the ability to selectively measure NO through the reaction of NO with oxygen plasma, giving
202 it the ability to reduce interference from molecules such as nitrates and nitrites40. The ability of
203 NOA to selectively measure NO has made this technique a gold standard in the field of NO-
204 releasing materials41-43. Films were punched to 5/16 diameter and threaded with silk surgical
205 suture to be suspended in an amber NOA cell. The NOA cell with the membrane is lowered into
206 a 37oC water bath while the amber cell protects the sample from light. Deionized water (3 mL)
207 was added to the NOA cell and allowed to heat to 37C. Nitrogen was bubbled into the DI at 100
9
208 mL/min to provide a humid environment for the film. Both 2-layer and 3-layer membranes
209 containing SNAP were tested for initial release, as well as after 24-hour storage in humid 37C
210 conditions in water jacketed incubator (Thermo Fisher Scientific, Waltham, MA USA). A
211 representation of the measurement cell is shown in Figure 1.
212 Zone of inhibition (ZOI) studies
213 Antibacterial properties of SNAP incorporated chitosan-nanocellulose based packaging
214 membranes were tested against three common bacterial strains using zone of inhibition (ZOI)
215 assay. The bacteria used in the study were Staphylococcus aureus (S. aureus), Listeria
216 monocytogenes (L. monocytogenes), and Enterococcus faecalis (E. faecalis). A modified
217 version of agar diffusion standard protocol was followed to carry out this study aseptically44, 45.
218 A single isolated colony of each bacterium was suspended individually in Luria broth (LB)
219 medium and allowed to grow at 37C for 14 hours at a rotating speed of 150 rpm using a shaker
220 incubator. The optical density (OD) of the liquid suspension of each of the strains was measured
221 by UV-Vis spectrophotometer (Genesis 10S-Thermo Scientific) at 600 nm (OD600) using LB
222 medium as blank and was adjusted to 1x107 colony forming units per mL (CFUs/mL) based on
223 the calibration curve between CFUs and OD. A sterile swab was placed into the bacterial
224 culture, gently pressed and rotated against the inside of the petridish (14 cm) to spread the
225 bacteria uniformly and aseptically. The circular pieces (dia= 14 mm) of packaging membranes
226 (control and test) were placed over the bacterial culture and pressed gently. The resulting plates
227 with bacterial strain and membranes were incubated overnight at 37C for 20h. The diameters of
228 the ZOI of the membranes were compared with each other and among the bacteria strains to
229 evaluate the antimicrobial effectiveness of the membranes.
230
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231 Results and Discussion
232 Water vapor permeability characteristics analysis
233 Table 1 shows the water vapor permeability of the control and SNAP incorporated packaging
234 membranes developed in this study. Control membranes prepared with a single material such as
235 chitosan or CNF showed less water vapor permeability compared to the other membranes.
236 Chitosan and CNF combined control membranes (2-layer and 3-layer) had slightly higher
237 permeability than the membranes with single material even though their thickness is slightly
238 higher than the single material control membranes. In ideal polymeric structures, gas and vapor
239 permeability are independent of film thickness46. However, the result from chitosan and CNF
240 membrane showed that it behaves like an ideal polymer. It is possible that during the
241 permeability test, the side exposed to high relative humidity absorbed more water and developed
242 desorption rate independent of the thickness-resistance for water vapor diffusion. As a result of
243 this, the side exposed at low relative humidity became responsible for the vapor transfer. Under
244 this condition, the diffusion flux could become independent of thickness and resulted in higher
245 permeability than the single component control film47. SNAP incorporated membranes had
246 higher water vapor permeability than the membranes prepared without SNAP. Even though the
247 thickness of SNAP incorporated membranes with 2-layer and 3-layer were significantly higher
248 than other control membranes, they demonstrated the highest permeability indicating that the
249 membranes containing SNAP had a role in increasing the water vapor permeability. However,
250 the water permeability values obtained in this study were lower than the methyl cellulose based
251 biodegradable membranes developed by Turhan and Sahbaz48 and other starch-based
252 biodegradable films made by Para et al49 and Bertizzi et al47. The addition of SNAP to the
253 chitosan cellulose matrix likely decreases the attractive forces between the chain networks hence
11
254 increasing the free volume and segmental motions, which could result in easy diffusion of water
255 molecules and thereby increase the water vapor permeability.
256 Tensile strength analysis
257 Figure 2 shows the Youngs modulus measured for control and NO-releasing membranes.
258 The mechanical strength of the membranes was studied by measuring their Young's modulus.
259 Since nanocellulose possess high mechanical strength, the membrane prepared with only
260 nanocellulose showed highest Young's modulus (1587.6 282 MPa) as compared to the other
261 membranes. Chitosan is structurally similar to cellulose except it contains NH2 group in the
262 position of the C2 hydroxyl group and mechanical properties of chitosan vary depending on the
263 percentage of deacetylation50. Chitosan used in this study was 85% deacetylated and its Youngs
264 modulus was measured at 245.4 89 MPa. The 2-layer and 3-layer control films showed higher
265 Young's modulus than control chitosan. As expected, chitosan and nanocellulose together
266 increased the mechanical strength due to increased hydrogen bonding between the polymer
267 chains of nanocellulose and chitosan51. However, incorporation of SNAP into the 2-layer and 3-
268 layer membranes reduced the mechanical strength much lower than the control chitosan films.
269 One of our previous studies showed similar reductions in strength upon incorporation of SNAP
270 into medical grade polymers, which can arise due to the solubility of the SNAP molecule within
271 the polymer matrix52. As the concentration of SNAP within the matrix surpasses the solubility
272 limit, localized regions of SNAP crystallization occur53. The localized crystalline SNAP might
273 create gaps between the nanocellulose network chains for its mobility and reduce inter-chain
274 interactions leading to significant reduction in tensile strength of the membrane. Similar to the
275 effect of SNAP on water vapor permeability, the addition of SNAP to the nanocellulose-chitosan
12
276 matrix might decrease the attractive forces between the network chains and increase the free
277 volume and segmental motions, which could cause a reduction in mechanical strength as well.
278 NO release characteristics
279 Nitric oxide released from the 2-layer and-3-layer chitosan compositions were measured
280 using the Sieves Chemiluminescence Nitric Oxide Analyzer (model 280i, Boulder, CO). The
281 release of nitric oxide (NO) from SNAP is highly sensitive to heat, light (340 and 590 nm), and
282 moisture, as these catalyze the spontaneous decomposition reaction54, 55. The 2-layer and 3-layer
283 designs were implemented to see if the top layer of chitosan-nanocellulose would provide a
284 barrier to help selectively deliver NO to one side of the film. This selective release of NO would
285 be accomplished by limiting the exposure of the SNAP layer to both light and moisture.
286 However, no significant difference was observed between the 2 and 3-layer configurations (p =
287 0.36) as measured using a two-tailed Student's t-test. Release of NO from the chitosan-
288 nanocellulose composites were measured for initial release, as well as a release after 24 hours to
289 determine the level of NO that is being delivered during zone of inhibition studies (Figure 3). All
290 measurements were conducted at 37C and the samples were protected from light at all times.
291 Initial release of NO from 2-layer and 3-layer composites were found to be 0.10.03x10-10
292 mol. cm-2. min-1 and 0.18 0.07 x 10-10 mol. cm-2. min-1 respectively. In both cases, release of
293 NO decreased after 24 hours at 37C to 0.07 0.01x10-10 mol. cm-2. min-1 and 0.05 0.01x10-10
294 mol. cm-2. min-1. While the NO release appears to be higher for the 3-layer configuration, it is
295 possible the NO release from the 2-layer configuration is asymmetric and is providing a larger
296 flux of NO to the exposed SNAP layer than that of the chitosan-nanocellulose layer. This could
297 result in an underestimation of the NO release.
298
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299 Antibacterial characteristics evaluation
300 Table 2 shows the zone of inhibition (ZOI) of each selected bacterium strain for both 2 and 3
301 layer membranes with NO-releasing (SNAP) component as an antimicrobial agent. The chitosan-
302 nanocellulose films with incorporated antimicrobial component resulted in the varying level of
303 NO release, which resulted in ZOI (mm) with varied diameters (Figure 4) depending on the level
304 of NO release. As expected all bacterial strains (S. aureus, L. monocytogenes, and E. faecalis)
305 were found susceptible to the packaging material owing to the bactericidal effect of NO. The
306 antimicrobial activity of the membranes resulted in similar ZOI between 2-layer and 3-layer
307 membranes against E. faecalis, and S. aureus. However, L. monocytogenes showed a significant
308 difference in the ZOI between the 2-layer and 3-layer membranes. Among all the bacteria, L.
309 monocytogenes was most susceptible to both the 3-layer and 2-layer membranes. Overall, S.
310 aurues exhibited the smallest ZOI (as compared to E. faecalis, and L. monocytogenes). The
311 higher antimicrobial activity of 3-layer membrane as compared to 2-layer membrane can directly
312 be correlated to the NO flux exhibited by these films. As shown in Figure 3, the 3-layer
313 membranes have higher NO flux in the beginning which might have resulted in higher bacteria
314 killing in the initial few hours. However, over an incubation period of 24 hours during ZOI
315 testing, the NO flux reached almost the same value for both 2-layer and 3-layer membranes
316 hence resulted in similar diameter of ZOI. The difference in the antimicrobial activity among the
317 bacterial strains could be attributed to their cell membrane properties56.
318 Morphology of membranes
319 Figure 5 shows the surface morphology of 2-layer and 3-layer control (A and B) membrane
320 and the membrane with SNAP material (C and D). Membranes were showed smooth surface
321 without any pores or cracks and with good structural integrity. The membranes obtained after
14
322 drying were flat and compact. All of them show the nanocellulose fibrils network structure. It
323 could be assumed that within the fibrils network chitosan was smoothly dispersed, because
324 chitosan disperses within the nanocellulose matrix with relatively good interfacial adhesion
325 between the two components50. These results could be attributed to the strong interactions
326 between nanocellulose fibrils and chitosan and SNAP, which are caused by the hydrogen
327 bonding between the hydroxyl groups of nanocellulose and carbonyl group in the chitosan.
328 In conclusion, S-nitroso-N-acetylpenicillamine incorporated antimicrobial membranes mixed
329 with nanocellulose fibrils and chitosan were successfully fabricated. Developed membranes
330 exhibited good film forming properties due to the presence of high density of amino groups and
331 hydroxyl groups and inter and intramolecular hydrogen bonding. The chitosan-CNF-NO-
332 releasing composites showed antimicrobial characteristics in the packaging membranes. The
333 addition of chitosan and SNAP into nanocellulose to develop composite biodegradable
334 membrane with antimicrobial activity showed clear effects towards inhibition of E. faecalis,
335 S.aureus, and L.monocytogenes as shown using ZOI. The membranes developed in this study
336 showed excellent water barrier property with a low value of water vapor permeability. Surface
337 morphology showed the strong interactions between nanocellulose fibrils, chitosan, and SNAP
338 materials. Tensile strength measurements showed decreased Youngs modulus for SNAP
339 incorporated membranes, which would be further studied to improve its mechanical properties.
340 Acknowledgement
341 Authors acknowledge the Poultry processing research unit in US poultry research center at
342 Athens, GA for proving the bacteria strains for this study.
343
344
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345 References
346 (1) U.S. Department of Health and Human Services and U.S. Department of Agriculture,
347 Dietary Guidelines for Americans, January 2005 (accessed on February 24th, 2016).
348 (2) U.S. Department of Agriculture and Centers for Disease Control and Prevention,
349 "MyPyramid, April 2005 (accessed on February 24th, 2016).
350 (3) U.S. Department of Agriculture, Economic Research Service, U.S. per Capita Food
351 Consumption of Fruits and Vegetables, 2005 (accessed on February 24th, 2016).
352 (4) Produce Marketing Association, "Fresh-cut Produce Industry" fact sheet, 2006. See web
353 site at http://www.pma.com/ (accessed on February 24th, 2016).
354 (5) Mead, P.S.; Slutsker, L.; Dietz, C. 2000. Food-Related Illness and Death in the United
355 States. J. Environ. Health. 2000, 62, 9-18.
356 (6) Allos, B.M.; Moore, M.R.; Griffin, P.M.; Tauxe, R.V. Surveillance for Sporadic
357 Foodborne Disease in the 21st Century: The Food-Net Perspective. Clin. Infect. Dis.
358 2004, 38(3), S115-120.
359 (7) Lampel, K.A.; Orlandi, P.A.; Kornegay, L. Improved Template Preparations for PCR-
360 Based Assays for Detection of Food-Borne Bacterial Pathogens. Appl. Environ.
361 Microbiol. 2000, 66(10), 4539-4542.
362 (8) Sivapalasingam, S.; Friedman, C.R.; Cohen, L.; Tauxe, R.V. Fresh Produce: A Growing
363 Cause of Outbreaks of Foodborne Illness in the United States, 1973 through 1997. J.
364 Food Prot. 2004, 67(10), 2342-2353.
365 (9) Tauxe, R.V. Emerging Foodborne Pathogens. Int. J. Food Microbiol. 2002, 78, 31-41.
16
366 (10) Trevejo, R.T; Courtney, J.G.; Starr, M.; Vugia, D.J. Epidemiology of
367 Salmonellosis In California, 19901999: Morbidity, Mortality, And Hospitalization
368 Costs. Am. J. Epidemiol. 2003,157:48-57.
369 (11) Leceta,I.; Gurrero,P.; Ibarburu,I.; Dueas,M.T.; Caba,K. Characterization And
370 Antimicrobial Analysis Of Chitosan-Based Films. J. Food Eng. 2013, 116, 889899.
371 (12) Sung, S.Y.; Sin, L.T.; Tee, T.T.; Bee, S.T.; Rahmat, A.R.; Vikhraman, M.
372 Antimicrobial Agents for Food Packaging Applications. Trends. Food Sci. Technol.
373 2013, 33, 110123.doi:10.1016/j.tifs.2013. 08.001
374 (13) Tre, H.; Gallstedt, M.; Hedenqvist, M.K. Antimicrobial Compression- Molded
375 Wheat Gluten Films Containing Potassium Sorbate. Food Res. Int. 2012, 45, 109115.
376 (14) Zhong, Y.; Song, X.; Li, Y. Antimicrobial, Physical And Mechanical Properties
377 Of Kudzu StarchChitosan Composite Films As A Function Of Acid Solvent Types.
378 Carbohydr. Polym. 2011, 84, 335342.
379 (15) Suppakul, P.; Miltz, J.; Sonneveld, K.; Bigger, S.W. Active Packaging
380 Technologies with an Emphasis on Antimicrobial Packaging and Its Applications. J.
381 Food. Sci. 2003, 68, 408420.
382 (16) Iwamoto, S., Abe, K.; Yano, H. The Effect of Hemicellulose on Wood Pulp
383 Nano-fibrillation and Nanofiber Network Characteristics. Biomacromolecules. 2008,
384 9:1022-1026.
385 (17) Suryanegara, L.; Nakagaito, A.N.; Yano, H. The Effect of Crystallization of PLA
386 on the Thermal and Mechanical Properties of Micro-fibrillated Cellulose-Reinforced
387 PLA Composites. Compos. Sci. Technol. 2009, 69:11871192.
17
388 (18) Nakagaito, A.N.; Fujimura, A.; Sakai, T.; Hama, Y.; Yano, H. Production of
389 Microfibrillated Cellulose (MFC)-Reinforced Polylactic Acid (PLA) Nanocomposites
390 From Stets Obtained By A Papermaking-Like Process. Comp. Sci. Techn. 2009, 69:1293
391 1297.
392 (19) Jonoobi, M.; Harun, J.; Mathew, A.P.; Oksman, K. Mechanical Properties of
393 Cellulose Nanofiber (CNF) Reinforced Polylactic Acid (PLA) Prepared By Twin Screw
394 Extrusion. Comp. Sci. Techn.. 2010, 70(12), 1742-1747.
395 (20) George, j.; Ramana, K. V.; Sabapathy, S. N.; Bawa, A. S. Physicomechanical
396 Properties of Chemically Treated Bacterial (Acetobacter Xylinum) Cellulose Membrane.
397 World J. Microb. Biot. 2005, 21, 13231327.
398 (21) Avik Khan.;, Tanzina Huq.; Ruhul A. Khan.; Bernard Riedl.; Monique Lacroix.
399 Nanocellulose-Based Composites and Bioactive Agents for Food Packaging. Crit. Rev.
400 Food Sci. Nutr. 2014, 54:163174.
401 (22) Dutta, P.K.; Shripra Tripathi; Mehrotra, G.K.; Joydeep Dutta. Perspectives for
402 Chitosan Based Antimicrobial Films in Food Applications. Food Chem. 2009, 114(4):
403 1173-1182.
404 (23) Shripra Tripathi; Mehrotra, G.K.; Dutta, P.K. Physicochemical and Bioactivity of
405 Crosslinked Chitosan-PVA Film for Food Packaging Applications. Int. J. Biol.
406 Macromolec. 2009, 45: 372-376.
407 (24) Shripra Tripathi; Mehrotra, G.K.; Dutta, P.K. Preparation and Physicochemical
408 Evaluation of Chitosan/PVA/Pectin Ternary Film for Food Packaging Applications.
409 Carbohydr. Polym. 2009, 79: 711-716.
18
410 (25) Debeaufort, F.; Voilley, A. Lipid Based Edible Films and Coatings, In Edible
411 Films and Coatings for Food Applications, M. E. Embuscado and K. C. Huber, Eds.,
412 2009, pp. 135168, Springer, New York, NY, USA.
413 (26) McHugh, T. H.; Avena-Bustillos, R. J. Applications Of Edible Films and
414 Coatings To Processed Foods, In Edible Coatings And Films to Improve Food Quality,
415 E. A. Baldwin, R. Hagenmaier, and J. Bai, Eds., 2012, pp. 291318, CRC Press, Boca
416 Raton, Fla, USA.
417 (27) Soliva-Fortuny, R.; Rojas-Graii, M.A.; Martin-Belloso, O. Polysaccharide
418 Coatings, In Edible Coatings and Films to Improve Food Quality, E. Baldwin, R.
419 Hagenmaier, and J. Bai, Eds., 2012, pp. 103136, CRC Press, Boca Raton, Fla, USA.
420 (28) Appendini, P.; Hotchkiss, J.H. Review of Antimicrobial Food Packaging.
421 Innovative Food Sci. Emerging. Technol. 2002, 3, 113126.
422 (29) Miranda, K. M.; Espey, M. G.; Jourd'heuil, D.; Grisham, M. B.; Fukuto, J. M.;
423 Feelisch, M.; Wink, D. A. The Chemical Biology of Nitric Oxide. In Ignarro, L.J. (ed.),
424 Nitric Oxide: Biology and Pathobiology, Academic Press, San Diego, California, 2000,
425 pp. 41-55.
426 (30) Cammack, R.; Joannou, C. L.; Cui, X. Y.; Martinez, C. T.; Maraj, S. R.; Hughes,
427 M. N. Nitrite And Nitrosyl Compounds in Food Preservation. Biochim. Biophys. Acta,
428 Bioenerg. 1999, 1411, 475-488.
429 (31) Kanner, J.; Juven, B. J. S-Nitrosocysteine as an Antioxidant, Color-Developing,
430 Andanticlostridial Agent in Comminuted Turkey Meat. J. Food. Sci. 1980, 45, 1105-
431 1108, 1112.
19
432 (32) De Groote, M. A.; Fang, F.C. NO Inhibitions: Antimicrobial Properties of Nitric
433 Oxide. Clin. Infect. Dis. 1995, 21 (2), S162-5.
434 (33) Jones, M.L.; Ganopolsky,J.G.; Labb, A.; Wahl, C.; Prakash, S. Antimicrobial
435 Properties of Nitric Oxide and its Application in Antimicrobial Formulations and Medical
436 Devices. Appl. Microbiol. Biotechnol. 2010, 88, 401407.
437 (34) Schairer, D.; Martinez, L.R.; Blecher, K.; Chouake, J.; Nacharaju, P.; Gialanella,
438 P.; Friedman, J.M.; Nosanchuk, J.D.; Friedman, A. J. Nitric Oxide Nanoparticles: Pre-
439 Clinical Utility as a Therapeutic For Intramuscular Abscesses. Virulence. 2012, 3, 6267.
440 (35) Nathan, C. Reactive Oxygen and Nitrogen Intermediates in the Relationship
441 between Mammalian Hosts and Microbial Pathogens. Proc Natl Acad .Sci. USA. 2000.
442 97, 8841-8.
443 (36) Privett, B. Examination of Bacterial Resistance to Exogenous Nitric Oxide. Nitric
444 Oxide. 2012. 26, 169-173.
445 (37) Friedman, A. Susceptibility of Gram-Positive and Negative Bacteria to Novel
446 Nitric Oxide-Releasing Nanoparticles. Virulence. 2011. 3(6), 45-50.
447 (38) Jaya, S.; Das, H. Accelerated Storage, Shelf Life and Color of Mango Powder. J.
448 Food Process. Preserv. 2005, 29, 4562.
449 (39) Labuza, T.P. Moisture Sorption: Practical Aspects of Isotherm Measurement and
450 Use. American Association of Cereal Chemists, 1984, Minnesota.
451 (40) Coneski, P.N; Schoenfisch, M.H. Nitric oxide release: part III. Measurement And
452 Reporting. Chem. Soc. Rev. 2012, 41, 3753-8.
20
453 (41) Brisbois, E.J.; Handa, H.; Major, T.C.; Bartlett, R.H.; Meyerhoff, M. E. Long-
454 Term Nitric Oxide Release and Elevated Temperature Stability with S-Nitroso-N-
455 Acetylpenicillamine (SNAP)-Doped Elast-Eon E2As Polymer. Biomaterials. 2013, 34,
456 6957-66.
457 (42) Handa, H.; Brisbois, E.J.; Major, T.C.; Refahiyat, L.; Amoako, K.A.; Annich,
458 G.M. In Vitro and In Vivo Study of Sustained Nitric Oxide Release Coating Using a
459 Diazeniumdiolate-Doped Poly (Vinyl Chloride) Matrix with the Poly (Lactide-Co-
460 Glycolide) Additive. J. Mater. Chem. B. 2013, 1, 3578-87.
461 (43) Handa, H.; Major, T.C.; Brisbois, E.J.; Amoako, K.A.; Meyerhoff, M.E.; Bartlett,
462 R.H. Hemocompatibility Comparison of Biomedical Grade Polymers Using Rabbit
463 Thrombogenicity Model For Preparing Nonthrombogenic Nitric Oxide Releasing
464 Surfaces. J. Mater. Chem. B. 2014, 2, 1059-67.
465 (44) Ericsson, H.; Tunevall, G.; Wickman, W. The Paper Disc Method for
466 Determination of Bacterial Sensitivity to Antibiotics: Relationship between the Diameter
467 of the Zone of Inhibition and the Minimum Inhibitory Concentration. Scand. J. Clin. Lab.
468 Invest. 1960, 12(4), 414-422.
469 (45) Rajni Singh; Astha Jain; Shikha Panwar; Deepti Gupta; Khare, S.K.
470 Antimicrobial Activity of some Natural Dyes. Dyes. Pigm. 2005, 66 (2), 99-102.
471 (46) Schwartzberg, H. G. Modeling of Gas and Vapor Transport through Hydrophilic
472 Films. In M. Mathlouthi (Ed.), Food packaging and preservation. Theory and practice,
473 1986, pp. 115136. London: Elsevier Applied Science Pub.
474 (47) Bertuzzi, M.A.; Castro Vidaurre, E.F.; Armada, M.; Gottifredi, J.C. Water Vapor
475 Permeability of Edible Starch Based Films. J. Food Eng. 2007, 80, 972978
21
476 (48) Nazan Turhan, K.; Ferhunde Sahbaz. Water Vapor Permeability, Tensile
477 Properties and Solubility of Methylcellulose-Based Edible Films. J. Food Eng. 2004, 61,
478 459466
479 (49) Parraa, D.F.; Tadinib, C.C.; Poncea, P.; Lugao. A.B. Mechanical Properties and
480 Water Vapor Transmission in Some Blends of Cassava Starch Edible Films. Carbohydr.
481 Polym. 2004, 58, 475481
482 (50) Wenling, C.; Duohui, J.; Jiamou, L.; Yandao, G.; Nanming, Z.; Xiufang, Z.
483 Effects of the Degree of Deacetylation on the Physicochemical Properties and Schwann
484 Cell Affinity of Chitosan Films. J. Biomater. Appl. 2005, 20(2), 157-77.
485 (51) Li, Z.; Zhuang, X. P.; Liu, X. F.; Guan, Y. L.; Yao, K. D. Study on Antibacterial
486 O-Carboxymethylated Chitosan/Cellulose Blend Film from Licl/N, N-
487 Dimethylacetamide Solution. Polymer. 2002, 43, 1541-1547.
488 (52) Goudie, M.J.; Brisbois, E.J.; Pant, J.; Thompson, A.; Potkay, J.A.; Handa, H.
489 Characterization of an S-Nitroso-N-Acetylpenicillamine-Based Nitric Oxide Releasing
490 Polymer from a Translational Perspective. Int. J. Polym. Mater. Biomater. 2016, Article
491 in press.
492 (53) Wo, Yaqi. Origin of Long-Term Storage Stability And Nitric Oxide Release
493 Behavior of Carbosil Polymer Doped with S-Nitroso-N-Acetyl-D-Penicillamine. ACS
494 Appl. Mater. Interfaces. 2015, 40(7), 22218-22227.
495 (54) Lyn H Williams, D. Identification of Cu+ as the Effective Reagent in Nitric
496 Oxide Formation from S-Nitrosothiols (RSNO). J. Am. Chem. Soc. 1996, 2 (4), 481-487.
22
515 (55) Frost, Megan C.; Mark E. Meyerhoff. Controlled Photo initiated Release of Nitric
516 Oxide from Polymer Films Containing S-Nitroso-N-Acetyl-DL-Penicillamine
517 Derivatized Fumed Silica Filler. J. Am. Chem. Soc. 2004, 126(5), 1348-1349.
518 (56) Herv Roy. Tuning the Properties of the Bacterial Membrane with Aminoacylated
519 Phosphatidyl Glycerol. IUBMB Life. 2009, 61(10), 940953.
520
521
522
523
524
525
526
527
528
Nitrogen Bubble flow
Suspension of NO releasing film

Nitrogen Sweep flow
NO rich gas to NOA
Water bath (37C)
529
23
530 Figure 1 Representation of test configuration for NOA cell for measurement of NO released
531 from chitosan films in a humid environment
532
533
534
535
2000
1800
1600
1400
Modulus (MPa)
1200
1000
800
600
400
200
0
2 layer control 3 layer control 2 layer NO 3 layer NO Cellulose Chitosan
control control
536
537 Figure 2 Youngs modulus of nanocellulose-chitosan membranes.
538
539
540
541
542
543
24
544
545
0.30
0.25
NO flux (x10-10 mol cm-2 min-1)
0.20
0.15
0.10
0.05
0.00
Chitosan 2 Layer (Initial) Chitosan 3 Layer (Initial) Chitosan 2 Layer (24 h) Chitosan 3 Layer (24 h)
546
547 Figure 3 Nitric oxide release rates of 2-layer and 3-layer chitosan-nanocellulose composites as
548 measured by chemiluminescence
549
550
551
552
553
554
25
555
556 Figure 4 Images of the zone of inhibition comparison of 2-layer chitosan-CNF control, 2-layer
557 SNAP incorporated chitosan-CNF film, 3-layer SNAP incorporated chitosan-CNF film and 3-
558 layer control (clockwise from top left) for (A) Listeria monocytogenes (B) Staphylococcus
559 aureus (C) Enterococcus faecalis.
560
561
562
563
564
565
26
566
567 Figure 5 Scanning electron microscope images of the chitosan-nanocellulose film before and
568 after addition of SNAP. A) 3-layer chitosan-nanocellulose, B) 2-layer chitosan-nanocellulose,
569 C) 3-layer chitosan-nanocellulose with SNAP, and D) 2-layer chitosan-nanocellulose with
570 SNAP.
571
572
573
574
575
576
577
578
579
580
581
27
582 Table 1. Water permeability of different types of packaging membranes
Membrane type Water vapor permeability Membrane
(kg.m/ m2.day.Pa) thickness (mm)
Chitosan 7.01x10-6 0.058
Nano Cellulose Fibrils (CNF) 6.88x10-6 0.060
2-layer CNF + Chitosan with SNAP 1.56x10-5 0.126
2-layer CNF +Chitosan without SNAP 8.81x10-6 0.068
3-layer CNF + Chitosan with SNAP 1.11x10-5 0.124
3-layer CNF + Chitosan without SNAP 8.31x10-6 0.096
583
584 Table 2. Comparative analysis of zone of inhibition among different bacterial strains using
585 developed antimicrobial packaging membranes
Film type Bacterial strain ZOI (mm)
2-layer SNAP incorporated chitosan-CNF films S. aureus 30
3-layer SNAP incorporated chitosan-CNF films S. aureus 30
2-layer SNAP incorporated chitosan-CNF films E. faecalis 34
3-layer SNAP incorporated chitosan-CNF films E. faecalis 35
2-layer SNAP incorporated chitosan-CNF films L. monocytogenes 37
3-layer SNAP incorporated chitosan-CNF films L. monocytogenes 45
586
587
588
589
28
590 Abstract Graphics
591
592 For table of contents only
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Antimicrobial and Physicochemical Characterization of Biodegradable, Nitric Oxide-Releasing Nanocellulose-Chitosan Packaging Membranes

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Antimicrobial and Physicochemical Characterization of Biodegradable, Nitric Oxide-Releasing Nanocellulose-Chitosan Packaging Membranes

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Journal of Agricultural and Food Chemistry is published by the American Chemical

1 Antimicrobial and Physicochemical Characterization of Biodegradable, Nitric Oxide-

2 Releasing Nanocellulose-Chitosan Packaging Membranes

5 School of Biological and Biochemical Engineering, College of Engineering, University of

6 Georgia, Athens, GA, USA

14 Telephone: (706) 542-3815

20 220 Riverbend Road

22 Telephone: (706) 542-8109

25 Biodegradable composite membranes with antimicrobial properties consisting of nanocellulose

31 Scanning Electron Microscopy (SEM) micrographs and chemiluminescence nitric oxide

35 evaluation of SNAP incorporated membranes showed an effective zone of inhibition against

36 bacterial strains of Enterococcus faecalis, Staphylococcus aureus, and Listeria monocytogenes

37 and demonstrated its potential applications for food packaging.

38 Keywords: nanocellulose fibrils; nitric oxide; SNAP; chitosan; antimicrobial; biodegradable;

39 packaging membranes; zone of inhibition

63 and detection of contaminations, enhanced epidemiological investigation, safe packaging, and

64 effective methods to identify pathogens5-10.

68 foodborne pathogens and food-spoiling microorganisms. These packages are typically

69 manufactured by incorporating antimicrobial agents, immobilized or coated on the surface of the

80 in terms of food safety, shelf-life, and environmental friendliness.

94 as a packaging material for the quality preservation of food22, 23, 24.

107 antibiotic-resistant strains35-37. However, incorporating NO donor compounds such as S-

109 investigated the effects of incorporating the NO donor, S-Nitroso-N-acetylpenicillamine (SNAP)

110 into biodegradable nanocellulose-chitosan composite membranes for increased antimicrobial

111 activity for potential food packaging application.

112 Materials and Methods

119 Synthesis of S-Nitroso-N-acetyl-D-penicillamine (SNAP)

129 Fabrication of composite membrane

147 time of 24 h; in total approximately 72 h to complete the processing of 3-layer configuration.

148 To prepare NO-releasing antimicrobial membranes, 2 wt % of chitosan was completely

161 CNF-chitosan mix without SNAP third.

162 Physicochemical characterization

163 Water permeability

173 following equation.

179 humidity (75%).

180 Tensile strength

191 material. All materials were tested in triplicate.

192 Morphology study using scanning electron microscopy

198 NO release measurements

211 representation of the measurement cell is shown in Figure 1.

212 Zone of inhibition (ZOI) studies

213 Antibacterial properties of SNAP incorporated chitosan-nanocellulose based packaging

221 by UV-Vis spectrophotometer (Genesis 10S-Thermo Scientific) at 600 nm (OD600) using LB

229 evaluate the antimicrobial effectiveness of the membranes.

231 Results and Discussion

232 Water vapor permeability characteristics analysis

255 molecules and thereby increase the water vapor permeability.

256 Tensile strength analysis

278 NO release characteristics

297 result in an underestimation of the NO release.

299 Antibacterial characteristics evaluation

317 bacterial strains could be attributed to their cell membrane properties56.

318 Morphology of membranes

328 In conclusion, S-nitroso-N-acetylpenicillamine incorporated antimicrobial membranes mixed

349 "MyPyramid, April 2005 (accessed on February 24th, 2016).

353 site at http://www.pma.com/ (accessed on February 24th, 2016).

355 States. J. Environ. Health. 2000, 62, 9-18.

358 2004, 38(3), S115-120.

361 Microbiol. 2000, 66(10), 4539-4542.

364 Food Prot. 2004, 67(10), 2342-2353.

367 Salmonellosis In California, 19901999: Morbidity, Mortality, And Hospitalization

368 Costs. Am. J. Epidemiol. 2003,157:48-57.

369 (11) Leceta,I.; Gurrero,P.; Ibarburu,I.; Dueas,M.T.; Caba,K. Characterization And