Antimicrobial and rheological properties of chitosan as affected by extracting conditions and humidity exposure

Accepted Manuscript Antimicrobial and rheological properties of chitosan as affected by extracting conditions and humidity exposure Mirari Y. Arancibia, M.Elvira López-Caballero, M.Carmen Gómez-Guillén, Marta Fernández-García, Fernando Fernández-Martín, Pilar Montero PII:

S0023-6438(14)00640-9

DOI:

10.1016/j.lwt.2014.10.019

Reference:

YFSTL 4216

To appear in:

LWT - Food Science and Technology

Received Date: 28 April 2014 Revised Date:

28 July 2014

Accepted Date: 5 October 2014

Please cite this article as: Arancibia, M.Y., López-Caballero, M.E., Gómez-Guillén, M.C., FernándezGarcía, M., Fernández-Martín, F., Montero, P., Antimicrobial and rheological properties of chitosan as affected by extracting conditions and humidity exposure, LWT - Food Science and Technology (2014), doi: 10.1016/j.lwt.2014.10.019. 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.

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Antimicrobial and rheological properties of chitosan as affected by extracting

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conditions and humidity exposure

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Mirari Y. Arancibiaa,b M. Elvira López-Caballeroa*, M. Carmen Gómez-Guilléna*, Marta

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Fernández-García3, Fernando Fernández-Martína, Pilar Monteroa

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a

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10. 28040 Madrid (Spain)

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Technical University of Ambato (UTA). Av. Los Chasquis y Río Payamino. Ambato (Ecuador).

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Instituto de Ciencia y Tecnología de Polímeros (ICTP-CSIC). C/ Juan de la Cierva 3, 28006

Institute of Food Science, Technology and Nutrition (ICTAN-CSIC**). C/ José Antonio Novais,

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Madrid, Spain

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* Corresponding author. Tel: +34-31-5492300; fax: +34-91-5493627.

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Email: [email protected]

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ACCEPTED MANUSCRIPT Abstract

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The antimicrobial activity of differently processed chitosans, of varying molecular weights

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(Mwv = 5600 – 690 kDa) and deacetylation degrees (DD = 77 – 86 %), was tested against 26

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microorganisms. Chitosan solutions and films were prepared by solubilizing chitosan with lactic

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acid without adding plasticizers. Films with different water activity (aw) were prepared by

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varying either drying time or relative humidity during film conditioning. In dilute solution (1

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g/100g, w/w), chitosan inhibited the growth of Gram-positive and Gram-negative bacteria, of

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which the most sensitive was Debaromyces hansenii. High antimicrobial activity was found in

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chitosans with different molecular weights and similar deacetylation degree (ChQ4: 1600 kDa,

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82 % DD; ChR2: 2200 kDa, 81 % DD; ChR4: 830 kDa, 83% DD). The films obtained from these

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chitosans were more effective when they were physically similar to coatings as a result of the

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increased aw, allowing diffusion of the active amino groups; moreover, this activity was further

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augmented at the more acidic pH. The superior rheological properties of the ChQ4 chitosan

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(1600 kDa, 82% DD) would also confer high mechanical resistance, which makes it a versatile

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option for the development of antimicrobial coatings to be used for a wide range of food

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applications.

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Key words: chitosan, water activity, antimicrobial, viscoelastic properties, film

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ACCEPTED MANUSCRIPT 1. Introduction

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Research on natural polymers has focused on developing more environmentally friendly

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packaging to reduce pollution caused by non-biodegradable material. In recent years, chitosan

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has aroused considerable interest in the industry because of its unique properties, including

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biodegradability, biocompatibility and non-toxicity (Dash, Chiellini, Ottenbrite, & Chiellini,

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2011).

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Chitosan solutions exhibit good film-forming capacity that makes them potentially useful for

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the development of antimicrobial coatings and films (Dutta, Tripathi, Mehrotra, & Dutta, 2009;

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Zhong, Song, & Li, 2011).The functional behaviour of chitosan may be related to its viscoelastic

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properties, which in turn, depends strongly on polymer concentration, temperature and

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molecular weight (Calero et al., 2010, Chattopadhyay & Inamdar, 2010). Since chitosan is a

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strong cationic polymer, highly adherent coatings can be formed, especially with high-viscosity

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chitosan solutions (Yamada et al., 2000). The viscoelastic properties of chitosan solutions have

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been positively correlated to their adherence capacity. Optimal coating adhesion would be the

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result of a balance between cohesiveness and macromolecular chain mobility, which would

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correspond to systems exhibiting an intermediate behaviour between a gel and a viscoelastic

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solution (Serrero et al., 2011).

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The antimicrobial activity of chitosan has been well recognized for many years, being

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considered a feasible alternative for bactericidal applications. The mechanism of action seems

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to be the result of a change in cell permeability produced by the chitosan charge and the

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surface characteristics of the bacterial cell wall (Devlieghere, Vermeulen, & Debevere, 2004).

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Some studies have related the antimicrobial effect of chitosan to the molecular weight (Mwv)

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and deacetylation degree (DD) (Zheng & Zhu, 2003). The pH and type of acid in which the

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chitosan is dissolved, as well as film storage conditions, can influence the antimicrobial

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properties (Bégin & Van Calsteren, 1999; Leceta, Guerrero, & de la Caba, 2013).

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Chitosan is produced mainly from the exoskeletons of crustaceans, basically by four steps:

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demineralization, deproteinization, decolourization and deacetylation. While this extraction

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process is widely used, Alvarado et al. (2007) developed a method in which the

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deproteinization and decolourization stages were obviated. The chitosan obtained in this way

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had a high molecular weight and a high deacetylation degree, as well as excellent film-forming

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ability compared to chitosans prepared following a conventional method. Moreover, the

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elimination of any stage reduces the cost of the process. Lalaleo (2010) reported a variation to

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ACCEPTED MANUSCRIPT the above method, whereby a reducing agent (NaBH4) was included in the deacetylation step,

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which lasted 2-4 hours. All these process modifications yielded chitosans with different

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molecular weights and deacetylation degrees; however, no information is available regarding

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their functional properties, particularly antimicrobial capacity and rheological behaviour.

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The aim of the present work was to study the functional behaviour of several differently

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processed chitosans by: i) determining the antimicrobial activity and viscoelastic properties in

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the form of dilute coating solutions, and ii) by comparing the antimicrobial capacity of chitosan

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coatings with that of the resulting films exposed to different relative humidity conditions.

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2. Materials and Methods

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2.1 Raw material

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Eight different chitosans were obtained from shrimp shells (Litopenaeus vannamei), following

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the methods described by Lalaleo (2010), with or without a prior chitin isolation step (Figure

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1). The chitosan (Ch) obtained by chitin prefetching consisted on shell deproteinization with

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0.5 g/100mL NaOH at 80 °C for 30 min and washing to neutral pH. A second deproteinization

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was carried out with 3 g/100mL NaOH for 10 min at 80 °C. This process was performed in

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triplicate. Samples were demineralized using 2 N HCl with a solid:liquid ratio of 1:3 (w/v) at

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ambient temperature for 60 min, and washed to neutral pH. The chitin thus obtained was

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washed to neutral pH and dried in a forced-air oven (FD 240 Binder, Tuttlingen, Alemania) for 6

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h at 50 °C. The chitin (Q) was first deacetylated using 50 g/100mL NaOH at 100 °C, following a

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slightly modified version of the method described by Alvarado et al. (2007), in the presence or

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absence of a reducing agent (R) (NaBH4) 0.83 g/L, with a deacetylation time of 2 or 4 h. The

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resulting chitosans were washed to neutral pH and dried for 6 h at 50 °C. For chitosan

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synthesis without the chitin step (Alvarado et al., 2007), the shrimp shells were demineralized

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and then deacetylated following the method described above. Table 1 summarizes the codes

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and extracting conditions for the different chitosans.

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2.2 Chitosan characterization

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2.2.1 Molecular weight

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The viscosity average molecular weight (Mwv) of chitosan was calculated from the

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experimental intrinsic viscosity [η] (mL/g) data using the Mark-Houwink-Sakurada-Staudinger

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equation

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[η] = K·(Mwv)α

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where K = 1.81×10-3 mL/g and α = 0.93 (Roberts & Domszy, 1982).

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Chitosan samples were carefully weighed and dissolved in 0.1 M acetic acid plus 0.2 M NaCl

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buffer solution. Variable amounts of the standard buffered chitosan solution and fresh buffer

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solution were used to prepare five diluted chitosan solutions at different concentrations.

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Relative viscosity was then measured with a Cannon-Fenske glass capillary viscometer at 25.0 ±

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0.1 °C. Reduced viscosity (ηred) was then calculated from the equation

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[ž] = lim žred

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as the intercept in the linear regression of viscosity versus concentration (C, g/dL).

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2.2.2 Deacetylation degree

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The deacetylation degree (DD) was determined by FTIR based on the ratio of absorbance (A) at

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1655 and 3450 cm-1 using the equation: DD = [1 – (A1655/A3450)/1.33]×100, following the

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method described by Kumirska et al. (2010). The FTIR spectra were recorded at room

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temperature using a Perkin Elmer Spectrum 400 Infrared Spectrometer (Perkin Elmer Inc.,

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Waltham, MA, USA) equipped with an attenuated total reflectance (ATR) prism crystal

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accessory. The spectra resolution was 4 cm-1 and 32 scans were averaged for each spectrum.

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All measurements were performed at least in triplicate and plotted as an average spectrum.

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Background subtraction was done using the Spectrum software version 6.3.2 (Perkin Elmer

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Inc.).

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2.2.3 Chitosan solubility

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To determine solubility, 100 mg of chitosan was mixed with 5 mL of 0.15 M lactic acid, stirred

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in a vortex mixer and allowed to stand for 24 hours. Complete solubility is characterized by the

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formation of a clear solution. The result was confirmed visually and was expressed as soluble

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chitosan: affirmation or denial.

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2.3 Preparation of chitosan solutions

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ACCEPTED MANUSCRIPT Solutions at concentrations of 1 g/100g (S1) or 3 g/100g (S3), used for determining

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antimicrobial properties and viscoelastic properties, respectively, were prepared by solubilizing

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chitosan in 0.15 M lactic acid (Panreac, Spain) with gentle stirring for 1 h at room temperature.

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The natural pH of the more dilute solution was 3.2 ± 0.2. The pH was corrected to 5.7 ± 0.2

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with 2 M NaOH (Panreac, Spain), and stirring continued for 24 h at 45 °C. During the stirring

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process, the container of the solution was covered with aluminium foil to prevent evaporation.

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To compare antimicrobial activity in a stronger acidic medium, selected chitosan solutions

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(ChQ4, ChR2 and ChR4) were prepared without pH correction.

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2.4 Preparation of chitosan films

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Films with different water activity were prepared by casting 50 g of chitosan solution (S1) in

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square plastic dishes (144 cm2) (Plexiglas® GS Röhm GmbH & Co. Kg, Darmstadt, Germany) and

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drying for 12 h (half-dried films) or 24 h (fully-dried films) at 45 ± 0.8 °C in a forced-air oven.

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Prior to analyses, the films were conditioned in a desiccator for 3d at 22 °C over saturated

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solutions of NaBr, NaCl and BaCl2 to provide relative humidity (RH) of 58 ± 0.2%, 75 ± 0.2% and

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90 ± 0.2% respectively.

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2.5 Water activity of films

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Water activity (aw) was measured at 25 °C using a LabMaster aw-meter (Novasina, Precisa,

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Poissy, France). All determinations were performed in triplicate.

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2.6 Antimicrobial properties

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The antimicrobial activity of the chitosan solutions and films was determined by the disk

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diffusion method in agar against 26 microbial strains as previously described (Gómez-Estaca,

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López de Lacey, López-Caballero, Gómez-Guillén, & Montero, 2010). Paper disks of 5 mm

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diameter soaked with the solution and 5 mm disks taken directly from the edible films were

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laid on the surface of BHI agar (Oxoid, Basingstoke, UK) plates previously inoculated with the

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microorganisms (105-106 CFU/mL). The strains, selected for their relevance to health (such as

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probiotics or pathogens) or as promoters of food spoilage, were obtained from the Spanish

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Standard Culture Collection (CECT): Aeromonas hydrophila CECT 839T, Aspergilus niger CECT

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2088, Bacillus cereus CECT 148, Bacillus coagulans CECT 56, Bifidobacterium animalis

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subespecie lactis DSMZ 10140, Bifidobacterium bifidum DSMZ 20215, Brochothrix

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thermosphacta CECT 847, Citrobacter freundii CECT 401, Clostridium perfringens CECT 486,

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Debaryomyces hansenii CECT 11364, Enterococcus faecium DSM 20477, Escherichia coli CECT

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ACCEPTED MANUSCRIPT 515, Lactobacillus acidophilus CETC 903, Lactobacillus helveticus DSM 20075, Listeria innocua

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CECT 910, Listeria monocytogenes CECT 4032, Penicilium expansum DSMZ 62841,

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Photobacterium phosphoreum CECT 4192, Pseudomonas aeruginosa CECT 110, Pseudomonas

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fluorescens CECT 4898, Salmonella cholerasuis CECT 4300, Shewanella putrefaciens CECT

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5346T, Shigella sonnei CECT 4887, Staphylococcus aureus CECT 240, Vibrio parahaemolyticus

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CECT 511T, Yersinia enterocolitica CECT 4315. After incubation, the inhibition diameter (taken

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as antimicrobial activity) was measured with Corel Photo-Paint X3 software. Results were

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expressed as diameter of growth inhibition (mm), including the diameter of the disk. Each

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determination was performed in duplicate.

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2.7 Viscoelastic properties of solutions

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Dynamic viscoelastic analysis of the chitosan solutions was carried out on a Bohlin CVO-100

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rheometer (Bohlin Instruments Ltd., Gloucestershire, UK) using a cone-plate geometry (cone

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angle 4°, gap 0.15 mm). Dynamic frequency sweeps were done at 5, 20 °C and 40 °C applying

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an oscillation amplitude of 0.2% in the frequency range 0.1-10 Hz. The elastic modulus (G’, Pa)

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and viscous modulus (G’’, Pa) were plotted as functions of frequency. The exponent n was

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calculated from the power law equation (G’=G0’ ωn).

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The dynamic temperature sweep was done by heating from 5 to 90 °C at a scan rate of 1

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°C/min, frequency 1 Hz and target strain 0.5%. The elastic modulus (G’; Pa) and viscous

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modulus (G’’; Pa) were plotted as functions of temperature in the heating ramp. Two

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determinations were performed for each sample, with an experimental error of less than 6% in

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all cases.

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2.8 Statistical analyses

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Statistical analyses were performed using analysis of variance (ANOVA). Tukey’s test was used

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in tables with GraphPad Prism 5.0 (GraphPad Software, San Diego California, USA). The level of

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significance was p ≤0.05.

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3. Results & Discussion

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The eight chitosans prepared in the present study passed the solubility test and had similar

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molecular weights and slightly higher degrees of deacetylation than those reported by Lalaleo

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(2010) (Table 1). The chitosans obtained without previous chitin isolation did not attain the 7

ACCEPTED MANUSCRIPT high DD (> 90%) observed by Alvarado et al. (2007), probably because different species were

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used. The addition of a reducing agent (NaBH4) protected the polymer chain, resulting in

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chitosans with slightly higher Mwv, especially with moderate deacetylation time (2h vs 4h), as

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reported by Lalaleo (2010). DD was only slightly affected by the different processing

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conditions, but Mwv was affected much more, decreasing with deacetylation time.

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3.1 Antimicrobial activity

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The antimicrobial activity of the eight types of chitosan was compared, initially in the form of

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diluted solutions at 1 g/100g (S1) (Table 2). All tested chitosans were effective against Gram

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positive and Gram negative microorganisms, of which B. coagulans was one of the most

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sensitive (p≤0.05). The sensitivity of bacteria to chitosan remains controversial, researchers

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arguing that Gram-positive (Jeon, Park and Kim, 2001) or Gram negative (Devlieghere et al.,

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2004) microorganisms are more sensitive to chitosan than the other. Probably the microbial

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strain can also influence the activity. For instance, in the present work the levels of inhibition

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of P. phosphoreum (G-) and E. faecium (G+) were similar. On the other hand, the highest

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observed level of inhibition was against D. hansenii, while the chitosan solutions were inactive

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against the moulds A. niger and P. expansum (p≤0.05) (Table 2). Roller et al., (2002) reported

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that yeasts were more sensitive than bacteria while chitosan appeared to be ineffective

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against fungi which contain chitosan as a cell wall component (Allan and Hadwinger, 1979).

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Results in the present experiment showed that all the solutions had antimicrobial activity,

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which differed considerably depending on the type of microorganism, rather than the type of

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chitosan. The antimicrobial activity of chitosan is related to physical status, type of

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microorganism, environmental factors, and intrinsic factors such as molecular weight and

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deacetylation degree (Kong, Chen, Xing, & Park, 2010). Antibacterial activity has been reported

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to be greater in low Mwv chitosan than in high Mwv chitosan (Kim, et al., 2011; Liu, et al.,

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2006). In addition, antimicrobial activity was found to be greater in highly deacetylated

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chitosans than in chitosan with a higher proportion of acetylated amino groups (Aider, 2010).

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This last author reports that a high deacetylation degree increases chitosan solubility and

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charge density, which are important factors for chitosan adhesion to the bacterial cell. In the

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present study, chitosan pairs with considerably different molecular weights, such as Ch2 and

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Ch4 or ChR2 and ChR4, showed similar levels of activity (Table 2), indicating that the molecular

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weight of chitosan was not a key factor for antimicrobial activity in the present work. In

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addition, results showed that despite enhancement of the degree of deacetylation of chitosans

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with time (e.g. from 81 to 83 for ChR2 and ChR4 respectively), this difference was not enough

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ACCEPTED MANUSCRIPT to influence its antimicrobial activity (Table 2). Park, Je, Byun, Moon, & Kim (2004) obtained

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chitosan with 75% DD in which antimicrobial activity was greater than in 90% or 50%

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deacetylated chitosan.

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In order to evaluate the influence of water activity on the capacity of chitosan to inhibit

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microorganisms, chitosans ChQ4, ChR2 and ChR4 were selected, based on their slightly higher

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activity against the bulk of microorganisms tested or against specific ones, as for instance

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ChQ4 against B. coagulans, D. hansenii or E. faecium (p ≤ 0.05) (Table 2). The selected chitosan

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solutions (ChQ4, ChR2 and ChR4) were dried for 12 h (half-drying time) and 24 h (full-drying

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time) to obtain films, which were conditioned at three relative humidity levels (58, 75 and 90

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% RH). Figure 2 shows the water activity (aw) of films as affected by drying time and relative

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humidity. There was no significant change in aw in the half-dried films as a function of relative

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humidity during conditioning, probably because not enough time elapsed for films to reach

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sorption equilibrium. Aw was noticeably higher in ChR4, the chitosan with the lowest molecular

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weight, probably because it was more hygroscopic. The fully-dried films conditioned at 58% RH

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showed much lower aw than the half-dried films irrespective of the chitosan type. However, in

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the case of the fully-dried films aw tended to rise with increasing RH. This effect was much

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more evident in the ChR4 chitosan film, in which after 3-days conditioning at 90 % RH, aw

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(0.94) was even higher than in the corresponding half-dried film (0.87).

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Irrespective of the chitosan, the antimicrobial activity was higher in half-dried films than in

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fully-dried films (p ≤ 0.05) since the gain of water content increased the total amount of active

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sites and improved the bactericidal effect of the chitosan (Tables 3, 4). In general, differences

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among the three types of chitosan films tested were almost negligible. In contrast to chitosan

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solutions, the antimicrobial activity of fully-dried films was much more limited to the area

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under the film in contact with the agar. This is because chitosan exerts its antimicrobial effect

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without the migration of active compounds, as reported by other authors (Brody, Strupinsky,

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and Kline, 2001; Leceta et al., 2013). Thus, as a solid matrix, chitosan appears to be trapped

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and its antimicrobial capacity is reduced (Zivanovic, Chi, & Draughon, 2005). Regarding the

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effect of RH, the largest inhibition halos were achieved with 90% RH; the effect was more

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pronounced in half-dried films (p ≤ 0.05) (Tables 3, 4), which in most cases presented the

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highest film water activity. In fact, in the present work, half-dried films exposed at 90% RH

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were quite similar in appearance to the high viscosity chitosan solutions (S3).

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ACCEPTED MANUSCRIPT The chitosan films were effective against microorganisms commonly associated with fish

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spoilage (e.g. S. putrefaciens, P. phosphoreum) and potential pathogens (e.g. L.

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monocytogenes, V, parahaemolyticus) (Tables 3, 4). If applied during chilled storage, these

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films could help prolong the stability of fish in an acceptable state, as reported for chitosan

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coatings applied on fish patties or fish sausages (López-Caballero, Gómez-Guillén, Pérez-

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Mateos, & Montero, 2005a; López-Caballero, Gómez-Guillén, Pérez-Mateos, & Montero,

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2005b).

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The activity of the original chitosan solution at pH 3.2 (with no pH correction) and its

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corresponding fully-dried film conditioned at 58 % RH was also evaluated against some of the

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mentioned microorganisms. The antimicrobial activity of chitosan films was enhanced at lower

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pH (data not shown), probably due to the increased proportion of free amino groups resulting

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from higher chitosan solubility (Vascónez, Flores, Campos, Alvarado, & Gerschenson, 2009; Li,

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Feng, & Yang, 2010). In addition, the antimicrobial activity was greater in the chitosan

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solutions than in the resulting films because of the lower availability of functional groups due

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to the decrease in the number of water molecules in the films.

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3.2 Viscoelastic properties of chitosan solutions

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The 1 g/100g (S1) (pH 5.7) chitosan solutions showed the typical behaviour of a dilute solution,

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with G’’>G’ (results not shown). This chitosan concentration was effective against the selected

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microorganisms and was also suitable for dipping treatment to produce a protective coating;

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however, at such a low concentration the dynamic oscillatory study results were too low to

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ensure accurate rheometer readings. The polymer concentration was then increased to 3% to

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gain enough entanglement density to study possible temperature-dependent sol-gel

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transitions and other thermal events associated with molecular weight or deacetylation

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degree.

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Figure 3 shows the viscoelasticity/temperature profiles of chitosan solutions (S3) at pH 5.7. At

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low and moderate temperatures (below 45°C), the behaviour of all samples was fluid-like, with

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G’ lower than G’’ (Chenite, Buschmann, Wang, Chaput, & Kandani, 2001; Tang, Du, Hu, Shi, &

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Kennedy, 2007). Such predominantly viscous behaviour was maintained over the whole

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temperature interval tested (5 - 90ᵒC) in the case of low molecular weight chitosans ChQR4,

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Ch4 and ChR4, where no cross-point (G’= G’’) was evidenced, indicating the prevalence of

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random-coil entanglement networks (Calero et al., 2010). These samples presented the lowest

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values of G’ and G’’ at low temperatures (< 10 °C) and also exhibited negative temperature

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of hydrogen-bonded polymer associations.

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Conversely, G’ values increased in samples ChQ2, ChQ4, ChQR2, Ch2 and ChR2 starting from

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temperatures which varied depending on the type of chitosan (namely 67°C, 82°C, 72°C, 67°C

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and 47°C respectively), promoting elastic behaviour where G’ > G’’. This effect, which was

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more pronounced in the case of ChQ2 chitosan, could be associated with the partial formation

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of chitosan clusters through more stable heat-induced hydrophobic interactions. At low

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temperatures, chitosan–water interactions protected the chitosan chains against aggregation.

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Polymer aggregation was clearly observable upon heating as water molecules were eliminated,

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allowing association of chitosan macromolecules (Chenite et al., 2000). Chitosan ChQ2

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presented the lowest DD (77%) and also relatively high Mwv (3000 kDa). Both characteristics

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made for high self-assembling capacity; however, the antimicrobial activity was not as high as

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with other chitosans tested. Interestingly, chitosan ChR2 exhibited relatively high thermal

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aggregation capacity, presumptively via hydrophobic interactions, despite the relatively low

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values of G’ and G’’ registered at low temperatures. This chitosan (ChR2), of medium

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molecular weight (2200 kDa, DD 81%), showed very high antimicrobial capacity when applied

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in solution. On the contrary, ChQ4 chitosan, which also showed good antimicrobial properties,

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displayed the opposite temperature-dependent behaviour. The lower molecular weight (1600

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kDa, DD 82%) in the case of ChQ4 could have contributed to considerably poorer thermal

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aggregation capacity than ChR2. Thus, molecular weight seems to be an important factor

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determining the contribution of hydrogen bonding and hydrophobic interactions to chitosan

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self-association phenomena as a function of temperature, however, no definite relationship

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between rheological and antimicrobial properties could be established. There was no evidence

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of a strong influence of DD, probably because the different chitosans were very similar in that

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respect.

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Selected chitosan solutions ChQ4, ChR2 and ChR4, which showed high antimicrobial activity

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and very different rheological behaviour, were further characterized by evaluating their

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frequency dependence at different temperatures. Figure 4 shows the mechanical spectra at 5,

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20 and 40 °C in terms of elastic modulus (G’) and viscous modulus (G’’) as a function of the

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angular frequency. The frequency dependence of G’ and G’’ varied considerably depending on

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the type of chitosan and the temperature. Chitosan ChQ4 presented noticeably higher G’ and

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G’’ over the whole frequency range, and was the only one where G’>G’’, regardless of

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temperature. The viscoelastic parameters derived from mechanical spectra, which had been

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ACCEPTED MANUSCRIPT calculated after fitting the power law, are also presented in Fig. 4. According to Zhou &

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Mulvaney (1998), G0’ and G0’’ indicate the resistance of the material, to elastic and viscous

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deformation respectively, at an angular frequency of 1rad/s. These parameters were

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considerably higher in ChQ4 than in ChR2 or ChR4 and tended to decrease as temperature

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increased. Similar temperature-dependent behaviour was also observed in the case of ChR2

316

and ChR4 chitosans, probably due to a heat-induced decrease of hydrogen bonded polymer

317

entanglements. The power law exponent n’ is related to structural conformation and stability

318

in the studied samples: the higher the n’ values, the greater is the instability of the matrix to

319

frequency changes. Samples ChR2 and ChR4, with average n’ values of ~1.24 and ~1.11

320

respectively, showed typical Newtonian liquid behaviour. On the contrary, ChQ4, with average

321

n’ values of ~0.62, followed the pattern reported by Scanlan & Winter (1991) (0.19-0.9), who

322

suggested that n’ values can vary depending on stoichiometry, polymer concentration and

323

molecular weight. Other authors consider that there is no universal value of n’, which is

324

probably related to the specific nature of each gelling system (Richter, Boyko, & Schröter,

325

2004). The viscoelastic behaviour of ChQ4 chitosan (1600 kDa, DD 82%) suggests the formation

326

of a more cohesive structural matrix, with large numbers of both intra- and intermolecular

327

interactions caused by hydrophobic interactions and hydrogen bonding, resulting in a more

328

complex system irrespective of the temperature. The main reason for the marked reduction of

329

G’ and G’’ observed in sample ChR4 (830 kDa; DD 83 %) may be that its molecular weight is

330

considerably lower. The molecular weight has been reported to be a main factor affecting the

331

viscosity of chitosan (Li et al., 2007; Klossner et al 2008). Thus, chitosan with lower molecular

332

weight would present less entangled junctions in solution, resulting in more flexible chains

333

which cause a reduction of viscosity. Nevertheless, factors other than Mwv and DD would have

334

to be involved to explain the intermediate rheological behaviour of the ChR2 chitosan (2200

335

kDa, DD 81%). Interestingly, this chitosan showed noticeably high thermal aggregation capacity

336

at high temperatures, which could be convenient for certain thermally processed food

337

preparations.

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4. Conclusion

340

Several chitosans with similar DD and different molecular weights were produced, all of them

341

showing noticeable antimicrobial properties when applied in the form of diluted solutions. The

342

antimicrobial capacity depended strongly on the type of microorganism, rather than the type

343

of chitosan. The resulting chitosan films exhibited antimicrobial activity in general limited to 12

ACCEPTED MANUSCRIPT the area in direct contact with the microorganisms. One way to improve the antimicrobial

345

activity of these films is to allow diffusion of chitosan active groups using partially dried films

346

or films maintained at high relative humidity levels with increased water activity (similar to a

347

coating but more viscous), in which there is greater availability and accessibility of positively

348

charged amino groups. No definite relationship could be established between molecular

349

weight, antimicrobial capacity and rheological behaviour. A conventionally extracted chitosan

350

with intermediate molecular characteristics, as is the case of ChQ4 (82% DD, 1600 kDa),

351

showed considerable antimicrobial activity and high self-assembling capacity, allowing the

352

formation of a cohesive structural matrix regardless of temperature. Thus, it represents a good

353

and versatile option for the development of antimicrobial coatings to be used for a wide range

354

of food applications. An alternative chitosan (ChR2: 81% DD, 2200 kDa) with reasonably good

355

antimicrobial and rheological properties was also produced without a prior chitin isolation

356

step, which is an advantage from an industrial point of view.

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Acknowledgements

359

This research was financed by the Spanish Ministry of Economy and Competitiveness through

360

project AGL2011-27607. Author M. Arancibia is funded by a SENESCYT Scholarship provided by

361

the Ecuadorian government.

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Figure legends

474

Figure 1. Scheme for obtaining different chitosans.

475 476

Figure 2. Water activity (aw) of (a) half-dried chitosan films and (b) fully-dried chitosan films

477

exposed at 58%, 75% and 90% relative humidity.

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Figure 3. Temperature dependence of elastic modulus (G’, Pa) and viscous modulus (G’’, Pa)

480

for chitosan solutions (S3) at pH 5.7 ± 0.2 upon heating from 5 to 90 °C.

481

Figure 4. Frequency dependence of elastic modulus (G’, Pa) and viscous modulus (G’’, Pa) for

483

chitosan solutions (S3) at pH 5.7±0.2: (a) at 5 °C, (b) 20 °C and (c) 40 °C.

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ACCEPTED MANUSCRIPT Table 1. Extracting conditions, Molecular weight (Mwv) and deacetylation degree (DD) of different chitosans prepared. Reducing agent

Deacetylation

isolation

NaBH4

Time (h)

DD

(kDa)

(%)

3000 1600 5600 1700 2100 690 2200 830

77 82 84 86 80 84 81 83

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ChQ2 Q 2 ChQ4 Q 4 ChQR2 Q R 2 ChQR4 Q R 4 Ch2 2 Ch4 4 ChR2 R 2 ChR4 R 4 Q: with previous chitin isolation; R: with reducing agent

Mwv

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Chitosan

Chitin

ACCEPTED MANUSCRIPT Table 2. Antimicrobial activity (mm) against selected microorganism of different chitosan solutions (1 g/100 g). Chitosan Microorganism

ChQ4

ChQR2

ChQR4

Ch2

Ch4

ChR2

ChR4

Aeromonas hydrophila

6 ± 0.01a

12 ± 0.03a,b,c

5 ± 0.01a

4 ± 0.06a

10 ± 0.01b,d

14 ± 0.01c

9 ± 0.03d

10 ± 0.74b,d

Aspergillus niger

5 ± 0.06a

4 ± 0.03a

5 ± 0.07a

5 ± 0.03a

5 ± 0.01a

5 ± 0.01a

5 ± 0.03a

7 ± 2,14a

Bacillus cereus

7 ± 0.08a,b

5 ± 0.03a

5 ± 0.08a

6 ± 0.03a

9 ± 0.03b

5 ± 0.01a

7 ± 0.04a,b

6 ± 1.4a

Bacillus coagulans

10 ± 0.06a,d

18 ± 0.03b

5 ± 0.03c

9 ± 0.06a

11 ± 0.04a,d,e

12 ± 0.03d,e

9 ± 0.04a

14 ± 0.39e

Bifidobacterium animalis

11 ± 0.14a,c

10 ± 0.04a

5 ± 0.04b

10 ± 0.06a

17 ± 0.03d

10 ± 0.04a

14 ± 0.06e

14 ± 0.66c,e

Bifidobacterium bifidum

8 ± 0.07a,c

7 ± 0.07a

8 ± 0.01a,c

12 ± 0.07b,d

12 ± 0.03b,d

12 ± 0.04b,d

10 ± 0.06b,c,d

11 ± 1.46d

5 ± 0.03a

5 ± 0.01a,c

4 ± 0.14a

6 ± 0.07a,b,c

7 ± 0.04b

5 ± 0.07a,b

7 ± 0.07b,c

6 ± 1.36a,b

11 ± 0.07c

8 ± 0.07b

11 ± 0.08c

12 ± 0.72c

8 ± 0.07b

5 ± 0.07a

5 ± 0.04a

5 ± 0.14a

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Brochothrix thermosphacta

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ChQ2

8 ± 0.01b

5 ± 0.14a

5 ± 0.06a

7 ± 0.04a,b

5 ± 0.08a

5 ± 0.06a

7 ± 0.01a,b

Debaryomyces hansenii

12 ± 0.03a

27 ± 0.11b

6 ± 0.11c,d

8 ± 0.01c,d,e

11 ± 0.06a,d

10 ± 0.08d,e

9 ± 0.03d

13 ± 0.09a

Enterococcus faecium

14 ± 0.08a

17 ± 0.10b

10 ± 0.08c

7 ± 0.01d

12 ± 0.06a,c

12 ± 0.11a,c,d

12 ± 0.01a,c

11 ± 1.3c

6 ± 0.14a,b

7 ± 0.03a,b

5 ± 0.04a

8 ± 0.03b

7 ± 1.99 a,b

7 ± 0.11b

16 ± 0.04c

13 ± 0.04c

14 ± 0.14c

14 ± 0.59c

5 ± 0.13b,c

8 ± 0.a01a,d

7 ± 0.03c

10 ± 0.04d

9 ± 2.02ca,c,d

8 ± 0.08a,b

7 ± 0.14a

5 ± 0.11b

7 ± 0.04a,b

7 ± 0.61a,b

Citrobacter freundii

7 ± 0.13a,b

5 ± 0.06a

8 ± 0.08a,b,c

10 ± 0.08a

6 ± 0.10b

Lactobacillus helveticus

6 ± 0.14a,b,c,d

8 ± 0.08a,c,d

4 ± 0.11b

Listeria monocytogenes

6 ± 0.01a,b

6 ± 0.07a,b

6 ± 0.11a,b

Listeria innocua

Escherichia coli

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5 ± 0.06a

Lactobacillus acidophilus

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5 ± 0.10a

Clostridium perfringens

10 ± 0.06a

3 ± 0.10b

5 ± 0.06b,c

7 ± 0.03c

6 ± 0.07c

7 ± 0.11c

7 ± 0.62c

5 ± 0.13a

4 ± 0.06a

5 ± 0.06a

5 ± 0.04a

4 ± 0.03a

5 ± 0.06a

5 ± 0.11a

5 ± 0.07a

Photobacterium phosphoreum

8 ± 0.11a

13 ± 0.04b

8 ± 0.07a

13 ± 0.08b

14 ± 0.06b

13 ± 0.06b

7 ± 0.11a

8 ± 1.5a

Pseudomonas aeruginosa

5 ± 0.07a,c

5 ± 0.04a,c

5 ± 0.06a,c

7 ± 0.14a,b

8 ± 0.06b

5 ± 0.06c

7 ± 0.06a,b,c

6 ± 1.37a,c

Pseudomonas fluorescens Salmonella cholerasuis

4 ± 0.03a 6 ± 0.04a,b

11 ± 0.11b,c 7 ± 0.14a,b

6 ± 0.08a 5 ± 0.01a

5 ± 0.14a 5 ± 0.11a

5 ± 0.04a 6 ± 0.08a,b

10 ± 0.14b,c 6 ± 0.08a,b

9 ± 0.10c 8 ± 0.06b

10 ± 0.74b,c 7 ± 2,11a,b

Shewanella putrefaciens

11 ± 0.07a,e

8 ± 0.14b,c

8 ± 0.03b,c

6 ± 0.03c

9 ± 0.08a,b

15 ± 0.01d

12 ± 0.10e

15 ± 3,61d,e

Shigella sonnei

5 ± 0.06a,c

5 ± 0.07a,c

5 ± 0.04a,c

7 ± 0.03a,c

10 ± 0.08b

6 ± 0.03c

11 ± 0.07d

12 ± 0.76b,d

Staphylococcus aureus

4 ± 0.03a

4 ± 0.06a

5 ± 0.03a,b,c

5.8 ± 0.04a,b

6 ± 0.10a,c

5 ± 0.03a,c

7 ± 0.10c

7 ± 0.65a,b,c

Vibrio parahaemolyticus

8 ± 0.08a

7 ± 0.08a

12 ± 0.11b,c

14 ± 0.01b

9 ± 0.11a

7 ± 0.04a

12 ± 0.10b,c

10 ± 3,45a,c

7 ± 0.08a,b,c,d

5 ± 0.11a

6 ± 0.10a,b,d

8 ± 0.01b,c

10 ± 0.04c,b,d

6 ± 0.08b

11 ± 0.14d

9 ± 3,51b,d

Yersinia enterocolitica

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8 ± 0.11a,c

Penicillum expansum

ACCEPTED MANUSCRIPT

Table 3. Antimicrobial activity (mm) against selected microorganisms of half-dried chitosan films conditioned at different relative humidity levels Microorganism

ChQ4

ChR2

ChR4

75

90

58

75

90

58

75

90

Aeromonas hydrophila

6.7 ± 0.08 a

7.7 ± 0.03b,e

10.2 ± 0.06 c

9.4 ± 0.05 d

7.7 ± 0.02 d

10.2 ± 0.04 d

7.7 ± 0.03 d

7.7 ± 0.14 f

8.6 ± 0.15 e

Aspergillus niger

10.2 ± 0.06 a

8.6 ± 0.02b,e

9.4 ± 0.03 a

10.2 ± 0.06 c

7.7 ± 0.03 d

9.4 ± 0.03 c

6.7 ± 0.06 f

8.6 ± 0.08 a

10.2 ± 0.09 e

Bacillus cereus

6.7 ± 0.04 a

6.7 ± 0.01 c

5.5 ± 0.00 b

5.5 ± 0.07 c

7.7 ± 0.03c,d

7.7 ± 0.10 a

6.7 ± 0.03 a

7.7 ± 0.03 c

13.4 ± 0.04 e

Bacillus coagulans

6.7 ± 0.05 a

6.7 ± 0.01 b

5.5 ± 0.04 a

6.7 ± 0.07 a

7.7 ± 0.03 c

8.6 ± 0.03 a

6.7 ± 0.04 a

6.7 ± 0.06a,b

6.7 ± 0.07 d

Bifidobacterium animalis

14.4 ± 0.09 a

6.7 ± 0.03 b

14.9 ± 0.03 c

10.2 ± 0.06 f

8.6 ± 0.03 e

10.2 ± 0.02 d

7.7 ± 0.02 d

9.4 ± 0.16gh

13.4 ± 0.18 g

Bifidobacterium bifidum

14.4 ± 0.09 a

10.2 ± 0.04b,c

12.8 ± 0.02 c

8.6 ± 0.08 f

9.4 ± 0.03 e

10.9 ± 0.02 d

7.7 ± 0.01 d

12.2 ± 0.17 h

10.9 ± 0.19 g

Brochothrix thermosphacta

7.7 ± 0.12 a

7.7 ± 0.05 c

10.2 ± 0.03 b

0.0 ± 0.00b,c

7.7 ± 0.03a,d

9.4 ± 0.02 a

7.7 ± 0.06 e

8.6 ± 0.23 c

8.6 ± 0.26 d

Citrobacter freundii

6.7 ± 0.09 a

6.7 ± 0.03c,d

6.7 ± 0.06 b

6.7 ± 0.15b,c,d

6.7 ± 0.06 a

7.7 ± 0.05 a

7.7 ± 0.03 d

8.6 ± 0.16 a

8.6 ± 0.18 a

Clostridium perfringens

13.4 ± 0.10 a

8.6 ± 0.04 b

9.4 ± 0.05 c

10.2 ± 0.03e,f

8.6 ± 0.10d,f

10.2 ± 0.04 d

7.7 ± 0.04 d

9.4 ± 0.18 g

12.8 ± 0.20f,g

Debaryomyces hansenii

8.6 ± 0.04 a

6.7 ± 0.01b,d

7.7 ± 0.05 c

10.2 ± 0.10d,e

8.6 ± 0.04 a

7.7 ± 0.04 c

6.7 ± 0.02 g

7.7 ± 0.04 f

6.7 ± 0.04b,e

Enterococcus faecium

6.7 ± 0.05 a

8.6 ± 0.02 b

7.7 ± 0.03c,h

7.7 ± 0.07 e

9.4 ± 0.03 d

7.7 ± 0.02 c

6.7 ± 0.01a,h

8.6 ± 0.07f,g

10.9 ± 0.08b,d,f

Escherichia coli

6.7 ± 0.00 a

7.7 ± 0.03 b

7.7 ± 0.01 c

8.6 ± 0.10d,e

7.7 ± 0.04 d

9.4 ± 0.01 d

7.7 ± 0.02d,e

7.7 ± 0.06 f

8.6 ± 0.06 e

Lactobacillus acidophilus

12.2 ± 0.05 a

7.7 ± 0.01 b

16.4 ± 0.00 c

9.4 ± 0.07d,h

8.6 ± 0.03 e

9.4 ± 0.00d,h

7.7 ± 0.01 h

7.7 ± 0.06 g

6.7 ± 0.06f,g

Lactobacillus helveticus

6.7 ± 0.03a,d,e

7.7 ± 0.00 a

6.7 ± 0.03 b

6.7 ± 0.07 c

7.7 ± 0.03 c

6.7 ± 0.02c,e

6.7 ± 0.03 e

7.7 ± 0.01a,d

10.2 ± 0.01 d 7.7 ± 0.16d,e

M AN U

SC

RI PT

58

RH (%)

6.7 ± 0.08 a

6.7 ± 0.03 b

8.6 ± 0.03 c

8.6 ± 0.06 c

7.7 ± 0.03 e

9.4 ± 0.02a,d

6.7 ± 0.00 a

7.7 ± 0.14 f

6.7 ± 0.07a,d

6.7 ± 0.03 c

6.7 ± 0.03 b

7.7 ± 0.08 d

6.7 ± 0.03 a

12.8 ± 0.02a,d

6.7 ± 0.01 d

6.7 ± 0.13 c

5.5 ± 0.14 a

Penicillum expansum

10.9 ± 0.13 a

6.7 ± 0.06 b

9.4 ± 0.03c,e

7.7 ± 0.08 d

6.7 ± 0.03 d

11.6 ± 0.03d,g

6.7 ± 0.01 d

6.7 ± 0.26 f

9.4 ± 0.29 e

Photobacterium phosphoreum

10.9 ± 0.04 a

6.7 ± 0.01 b

8.6 ± 0.04c,f

7.7 ± 0.08 cd

8.6 ± 0.04 e

9.4 ± 0.03 d

6.7 ± 0.06 f

8.6 ± 0.04 d

8.6 ± 0.04d,e,f

Pseudomonas aeruginosa

6.7 ± 0.10 a

7.7 ± 0.04 b

Pseudomonas fluorescens

8.6 ± 0.06 a

6.7 ± 0.02 c

Salmonella cholerasuis

6.7 ± 0.06 a

7.7 ± 0.02 a

Shewanella putrefaciens

7.7 ± 0.07 a

7.7 ± 0.03 b

Shigella sonnei

5.5 ± 0.02 a

Staphylococcus aureus

6.7 ± 0.12 a

Vibrio parahaemolyticus

7.7 ± 0.10 a

Yersinia enterocolitica

6.7 ± 0.10 a

TE D

Listeria monocytogenes Listeria innocua

7.7 ± 0.07 d

7.7 ± 0.03d,e

10.2 ± 0.04 d

6.7 ± 0.04 a

7.7 ± 0.19 f

11.6 ± 0.21 e

7.7 ± 0.07 a

7.7 ± 0.03 d

6.7 ± 0.03 c

5.5 ± 0.04 c

8.6 ± 0.09 c

16.8 ± 0.10 e

9.4 ± 0.04 b

8.6 ± 0.06b,c

8.6 ± 0.03 d

6.7 ± 0.03 c

6.7 ± 0.05 a

7.7 ± 0.10 f

7.7 ± 0.11 e

11.6 ± 0.05 c

8.6 ± 0.08 d

8.6 ± 0.03 e

9.4 ± 0.04a,c

7.7 ± 0.04 a

8.6 ± 0.12 f

10.2 ± 0.13 d

6.7 ± 0.02 b

8.6 ± 0.06 c

8.6 ± 0.12 g

7.7 ± 0.01b,e

7.7 ± 0.05 d

6.7 ± 0.02 d

8.6 ± 0.06 h

10.2 ± 0.03 g

7.7 ± 0.05 a

8.6 ± 0.05 b

7.7 ± 0.10 c

7.7 ± 0.04 c

6.7 ± 0.04 c

6.7 ± 0.01 a

7.7 ± 0.23 e

10.2 ± 0.26 d

7.7 ± 0.04 b

10.2 ± 0.05 c

9.4 ± 0.07 e

7.7 ± 0.03 a

10.2 ± 0.04a,d

6.7 ± 0.02 f

7.7 ± 0.19 d

18.5 ± 0.21b,d,e

6.7 ± 0.02 b

5.5 ± 0.10 d

7.7 ± 0.04 c

7.7 ± 0.02 c

6.7 ± 0.01 a

6.7 ± 0.19 c

11.6 ± 0.21 a,d

AC C

EP

7.7 ± 0.05 c

9.4 ± 0.03 b

7.7 ± 0.04 c

Different letters in the same row (a, b, c, d) indicate significant differences

ACCEPTED MANUSCRIPT

Table 4. Antimicrobial activity (mm) against selected microorganisms of full-dried chitosan films conditioned at different relative humidity levels Microorganism RH (%)

ChQ4

ChR2

ChR4

75

90

58

75

90

58

75

90

Aeromonas hydrophila

7.7 ± 0.03b

7.7 ± 0.01b,d

6.7 ± 0.02a

7.7 ± 0.01b

7.7 ± 0.02b

7.7 ± 0.02b,d

7.7 ± 0.07d

8.6 ± 0.02c

6.7 ± 0.02a

Aspergillus niger

6.7 ± 0.05b

7.7 ± 0.01a,c,d

6.7 ± 0.01 a

7.7 ± 0.03e

9.4 ± 0.08d,e,f

8.6 ± 0.01c

9.4 ± 0.13e

10.2 ± 0.01f

9.4 ± 0.01b

Bacillus cereus

6.7 ± 0.01b

6.7 ± 0.00a,b

6.7 ± 0.02 a

6.7 ± 0.00a,b

6.7 ± 0.00a,b

7.7 ± 0.01c

6.7 ± 0.06d

7.7 ± 0.00c

7.7 ± 0.00c

Bacillus coagulans

6.7 ± 0.01c

7.7 ± 0.01b

10.2 ± 0.02 a

7.7 ± 0.00b

7.7 ± 0.00b

6.7 ± 0.01c,d

6.7 ± 0.08d

7.7 ± 0.01b

10.9 ± 0.01c

Bifidobacterium animalis

7.7 ± 0.10c,d,e,f

8.6 ± 0.01b

12.2 ± 0.02 a

9.4 ± 0.09d,e

6.7 ± 0.01d

16.4 ± 0.02c

7.7 ± 0.04b

10.2 ± 0.02f

14.4 ± 0.01c

Bifidobacterium bifidum

7.7 ± 0.02c,d,e,f

8.6 ± 0.01b

14.9 ± 0.01 a

10.2 ± 0.00c,e

7.7 ± 0.01d

10.2 ± 0.02c,d

8.6 ± 0.01c

10.2 ± 0.02f

12.8 ± 0.01c

7.7 ± 0.03c,d

9.4 ± 0.02b

6.7 ± 0.01 a

9.4 ± 0.01b

7.7 ± 0.01d

8.6 ± 0.03c

8.6 ± 0.13d,e

8.6 ± 0.03c

7.7 ± 0.02c,d

6.7 ± 0.02b

8.6 ± 0.01a,d

6.7 ± 0.02a,b,c

6.7 ± 0.01b

6.7 ± 0.03b

7.7 ± 0.10c,d,e

8.6 ± 0.02b

8.6 ± 0.01 a

9.4 ± 0.09d,e

Clostridium perfringens Debaryomyces hansenii Enterococcus faecium

5.5 ± 0.00c

6.7 ± 0.00b,d

14.9 ± 0.01 a

6.7 ± 0.01d

6.7 ± 0.02a,c

7.7 ± 0.01b,d,e

7.7 ± 0.02a,d,e

7.7 ± 0.00d

6.7 ± 0.02b

7.7 ± 0.06d

7.7 ± 0.02e

8.6 ± 0.01d

7.7 ± 0.02d

6.7 ± 0.02c

10.2 ± 0.08d

10.9 ± 0.03e

8.6 ± 0.02a,b

SC

Citrobacter freundii

6.7 ± 0.02b

18.9 ± 0.01c,d

7.7 ± 0.07d

7.7 ± 0.01e

13.9 ± 0.00c

6.7 ± 0.01c,d

7.7 ± 0.01a,b

6.7 ± 0.01d

8.6 ± 0.01c

10.2 ± 0.01e

M AN U

Brochothrix thermosphacta

RI PT

58

Escherichia coli

6.7 ± 0.02b

5.5 ± 0.04a

6.7 ± 0.02a,b,c,d

8.6 ± 0.01c

6.7 ± 0.01b

6.7 ± 0.00b,d

6.7 ± 0.04d,e

7.7 ± 0.02d

9.4 ± 0.01d

Lactobacillus acidophilus

6.7 ± 0.00b

6.7 ± 0.08b

7.7 ± 0.01 a

8.6 ± 0.01e

6.7 ± 0.01b,d

8.6 ± 0.01c,d

9.4 ± 0.06e

10.2 ± 0.01c

9.4 ± 0.00c

Lactobacillus helveticus

6.7 ± 0.05b,d

6.7 ± 0.03b

7.7 ± 0.02a,d

5.5 ± 0.04c

7.7 ± 0.01a,c,d

6.7 ± 0.01b

8.6 ± 0.05c

7.7 ± 0.00d

6.7 ± 0.07c

Listeria monocytogenes

6.7 ± 0.02b,e

8.6 ± 0.01a,c

6.7 ± 0.02a,e

6.7 ± 0.00e

7.7 ± 0.0d1

8.6 ± 0.02c

8.6 ± 0.01c

7.7 ± 0.02d

8.6 ± 0.01b

6.7 ± 0.00c

7.7 ± 0.01b,d

5.5 ± 0.02 a

7.7 ± 0.01d

7.7 ± 0.01b,d

6.7 ± 0.02c

6.7 ± 0.03c

6.7 ± 0.02e

5.5 ± 0.01c

Listeria innocua

9.4 ± 0.02b

7.7 ± 0.06 a

8.6 ± 0.02d

6.7 ± 0.01c

9.4 ± 0.03b

7.7 ± 0.01a

5.5 ± 0.04e

7.7 ± 0.02a

10.2 ± 0.03b

6.7 ± 0.00a,d

10.2 ± 0.02 a

5.5 ± 0.01c

6.7 ± 0.01d

10.9 ± 0.01c

8.6 ± 0.13d

7.7 ± 0.01c

14.4 ± 0.00b

Pseudomonas aeruginosa

6.7 ± 0.01c,d

8.6 ± 0.02b

Pseudomonas fluorescens

6.7 ± 0.02c

7.7 ± 0.01b

Salmonella cholerasuis

6.7 ± 0.02b

7.7 ± 0.01a,c

Shewanella putrefaciens

6.7 ± 0.05c,e

7.7 ± 0.01b

Shigella sonnei

6.7 ± 0.05b,d

6.7 ± 0.01b,d

Staphylococcus aureus

7.7 ± 0.06b,c

Vibrio parahaemolyticus

9.4 ± 0.01a,e 8.6 ± 0.07b

6.7 ± 0.00d

7.7 ± 0.04a

6.7 ± 0.02c,d

7.7 ± 0.09a

7.7 ± 0.03a

7.7 ± 0.02a

8.6 ± 0.00d

5.5 ± 0.02a

7.7 ± 0.01b

7.7 ± 0.09b

8.6 ± 0.01d

7.7 ± 0.01b

6.7 ± 0.01a,b,d

9.4 ± 0.00d

7.7 ± 0.03c

6.7 ± 0.01b,d

6.7 ± 0.01d

9.4 ± 0.01b

6.7 ± 0.01b

7.7 ± 0.01 a

6.7 ± 0.01c

6.7 ± 0.02c

8.6 ± 0.02d

9.4 ± 0.07d,e

9.4 ± 0.02e

8.6 ± 0.01e

7.7 ± 0.01 a

6.7 ± 0.04d

6.7 ± 0.03b,d

8.6 ± 0.01c

7.7 ± 0.04a

7.7 ± 0.01a

8.6 ± 0.01c

9.4 ± 0.02a

7.7 ± 0.02a,c,d,e

7.7 ± 0.04c

6.7 ± 0.02d,e

6.7 ± 0.03c,d

7.7 ± 0.01e

6.7 ± 0.03d

7.7 ± 0.02c

8.6 ± 0.02b

6.7 ± 0.02a,c,e

3.9 ± 0.01d

6.7 ± 0.02c,e

6.7 ± 0.02c

8.6 ± 0.04b

7.7 ± 0.03d

6.7 ± 0.02e

7.7 ± 0.02 a

6.7 ± 0.06c

6.7 ± 0.01d

5.5 ± 0.02c

6.7 ± 0.01d

7.7 ± 0.03a

6.7 ± 0.02a

EP

7.7 ± 0.02 a

5.5 ± 0.01 a

AC C

Yersinia enterocolitica

TE D

6.7 ± 0.04c

Photobacterium phosphoreum

Penicillum expansum

8.6 ± 0.02b

Different letters in the same row (a, b, c, d) indicate significant differences

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

Figure 1

ACCEPTED MANUSCRIPT

(a) 1.0

RI PT

aw

0.9 0.8

58

75

SC

0.7

90

ChQ4

ChR2

M AN U

Relative Humidity (%)

ChR4

(b)

1.0

TE D

aw

0.8

0.6

EP

0.4

58

75

90

AC C

Relative Humidity (%)

ChR2

Figure 2

ChQ4

ChR4

ACCEPTED MANUSCRIPT 10000

1000

(a)

RI PT

G´(Pa)

100

10

SC

1

0.01 0

20

100

80

(b)

AC C 0.01 40

60

Temperature (°C)

FIGURE 3

80

Ch2

*

0.1

20

ChQR4

Ch4 ChR2

1

0

ChQR2

100

EP

G" (Pa)

10

40 60(°C) Temperature

ChQ4

+

TE D

1000

M AN U

0.1

ChQ2

100

ChR4

ACCEPTED MANUSCRIPT 1000

(a)

1000 G0’= 76.08

(d)

n’ = 0.52

G0’’= 73.88

n’’ = 0.42 n’’ = 0.84

G0’’= 10.42

n’’ = 0.86

100

100 n’ = 1.14 n’ = 1.01

1 G0’= 0.81

10

G0’’= 4.38

1

0.1

0.1

0.01

0.01

RI PT

G0’= 2.33

G" (Pa)

0.001

0.001 1

0

10

1000

Frequency (Hz)

100

G0’= 31.69

(b)

1000

n’ = 0.58

100

1

Frequency (Hz)

M AN U

0

SC

G´ (Pa)

10

10

(e)

G0’’= 35.498

n’’ = 0.48 n’’ = 0.90

G0’’= 6.84

n’’ = 0.96

n’ = 1.18

10

1 0.1

G" (Pa)

G0’= 1.04

n’ = 1.10

0.001 0

1

TE D

0.01

EP

100

(c)

n’ = 0.77

0.1

G´(Pa)

AC C

G0’= 9.49

1

G0’’= 1.82

n’ = 1.18

G0’= 0.39 n’ = 1.22

0.001 0

1

10

Frequency (Hz)

1000

(f)

100 10

G0’’= 15.87

n’’ = 0.61 n’’ = 0.97

G0’’= 3.29

n’’ = 1.01

1 G0’’= 1.09 0.1

G0’= 0.06

0.01

0.01

10

Frequency (Hz)

1000

10

1

0.1

G0’= 0.21

G" (Pa)

G´(Pa)

10

0.01

0.001

0.001 0

1

10

0

Frequency (Hz)

FIGURE 4

1 Frequency (Hz)

ChQ4

ChR2

ChR4

10

ACCEPTED MANUSCRIPT HIGHLIGHTS Antimicrobial and rheological properties of chitosan solutions were not related Rheological behavior was affected by the molecular weight of chitosan The antimicrobial capacity depended strongly on the type of microorganism Chitosan films with increased Aw improved their antimicrobial properties A functional chitosan without previous chitin isolation step was obtained

AC C

EP

TE D

M AN U

SC

RI PT

• • • • •