Tailoring physicochemical properties of chitosan films and their protective effects on meat by varying drying temperature

Accepted Manuscript Title: Tailoring physicochemical properties of chitosan films and their protective effects on meat by varying drying temperature Authors: Fei Liu, Wei Chang, Maoshen Chen, Feifei Xu, Jianguo Ma, Fang Zhong PII: DOI: Reference:

S0144-8617(19)30161-4 https://doi.org/10.1016/j.carbpol.2019.02.019 CARP 14592

To appear in: Received date: Revised date: Accepted date:

21 November 2018 1 February 2019 6 February 2019

Please cite this article as: Liu F, Chang W, Chen M, Xu F, Ma J, Zhong F, Tailoring physicochemical properties of chitosan films and their protective effects on meat by varying drying temperature, Carbohydrate Polymers (2019), https://doi.org/10.1016/j.carbpol.2019.02.019 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.

Tailoring physicochemical properties of chitosan films and their protective effects on meat by varying drying temperature

State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi

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Fei Liua,b, Wei Changa,b, Maoshen Chena,b, Feifei Xua,b, Jianguo Maa,b, Fang Zhonga,b,*,

214122, China.

School of Food Science and Technology, Jiangnan University, Wuxi 214122, China

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*Corresponding author: Fang Zhong

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Tel: +86-510-85197876, E-mail: [email protected]

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Graphical Abstract

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Highlights

Chitosan films dried at higher temperatures showed smoother micromorphology.

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Less ordered structures with smaller sizes were formed within chitosan films

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Higher drying temperatures led to inferior mechanical and barrier properties of

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dried at higher temperatures.

chitosan films.

Meat wrapped with chitosan films maintained the freshness for longer times than

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that with low density polyethylene (LDPE) film. Preservation effects of chitosan films on chilled meat decreased with increasing

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drying temperature. Abstract A higher temperature is usually used to increase the evaporation rate and thus reduce 2

the drying time of chitosan films during casting preparation process. The effects of drying temperature (45-85 °C) on the microstructure, mechanical and barrier properties of chitosan films were investigated. Chitosan films dried at higher temperatures showed smoother internal microstructures by forming smaller

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micro-region aggregations and lower ordered crystalline structures. Higher drying

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temperature also decreased the intermolecular interactions of chitosan chains

according to Fourier transform infrared spectroscopy analysis. These together led to the decrease in tensile strength, and the increase in water vapor and oxygen

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permeability. The film surface hydrophobicity remained unchanged at different drying

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temperatures, suggesting the applicability for chilled meat preservation. Chilled meat

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packaged with chitosan films had appropriate drip loss rate values as compared to those of the low-density polyethylene film and the blank sample, with lower

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thiobarbituric acid reactive substances, aerobic plate count, pH, and total volatile

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basic nitrogen values during 10 d storage. Moreover, films dried at lower

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temperatures showed superior juice retention capacity as well as superior preservation effect on chilled meat. The results found in this study can be used to better guide the selecting of drying temperature for chitosan film preparation.

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Keywords: Chitosan films; drying temperature; microstructure; mechanical and barrier properties; chilled meat.

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1. Introduction Recently, more attention has been focused on the natural biopolymer packaging materials, which are environmentally friendly and naturally biodegradable (Srinivasa,

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Ramesh, Kumar & Tharanathan, 2004). Among these natural biopolymers, chitosan is one of the most promising polymers for biodegradable packages. It is a

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polysaccharide derived from chitin by N-deacetylation with strong alkali, which is abundant available in crustacean wastes (Srinivasa, Ramesh, Kumar & Tharanathan, 2004).

Chitosan

of

consists

β-(1-4)-2-acetamido-D-glucose

and

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β-(1-4)-2-amino-D-glucose units (Sun, Sun, Chen, Niu, Yang & Guo, 2017). Its chain

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structure endows it with a good film-formation property, which has been reported by

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many studies (Jahit, Nazmi, Isa & Sarbon, 2016). Chitosan films had comparable

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mechanical and oxygen barrier property properties to various medium-strength synthetic polymers (Fernández-Pan, Ziani, Pedroza-Islas & Maté, 2010). The inherent

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antimicrobial property further makes chitosan as an ideal biodegradable packaging

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material (Aider, 2010; Butler, Vergano, Testin, Bunn & Wiles, 1996). Casting solvent evaporation technique is the main method usually used for the

preparation of biopolymer films (Mayachiew & Devahastin, 2010; Priyadarshi, Sauraj,

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Kumar & Negi, 2018). However, the casting technique limits the film preparation in industry due to the long drying times. A high drying temperature is often used to elevate the evaporation rate and thus shorten the drying time (Kayserilioǧlu, Bakir, Yilmaz & Akkaş, 2003; Soazo, Rubiolo & Verdini, 2011). The degree of 4

reorganization or crystallinity of the film may be determined by the drying temperature, thus affecting the mechanical and barrier properties of films (Homez-Jara, Daza, Aguirre, Munoz, Solanilla & Vaquiro, 2018; Silva, Bierhalz & Kieckbusch, 2012).

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However, contrary results were previously reported about the influence of drying

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temperature on the film properties. For instance, Alcantara, Rumsey and Krochta

(1998) reported that increasing the drying temperature could improve the mechanical and barrier properties of whey protein isolate films. But, Silva, Bierhalz and

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Kieckbusch (2012) showed that higher drying temperatures caused a decrease in film

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tensile strength and barrier properties of alginate film. In the case of chitosan film,

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Fernández-Pan, Ziani, Pedroza-Islas and Maté (2010) also reported that the performance of chitosan films was more susceptible to the drying temperature, where

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higher drying temperatures led to lower tensile strength and water vapor permeability

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values. These contrary results might be resulted from the differences in biopolymer

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structures and their film microstructures after drying at different temperatures. The purpose of this study is to analyze the effect of drying temperature on the

microstructure, mechanical and barrier properties of chitosan films, and thus to

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investigate the relationship among these properties. Moreover, chitosan films were also tested with regard to the preservation effects on chilled meat. Meat is susceptible to oxidative deterioration and microbial spoilage, resulting in sensory changes (flavors and aromas), loss of nutrients (essential fatty acids and fat-soluble vitamins), 5

and even health hazards (Ajuyah, Fenton, Hardin & Sim, 1993; Cai, Wu, Li, Zhong, Li & Li, 2014; Sánchezortega, Garcíaalmendárez, Santoslópez, Amaroreyes, Barbozacorona & Regalado, 2014). Although the delay in bacterial damage and improvement in sensory acceptance of meats packaged by chitosan films has been

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previously reported, the influences of drying temperature on their preservation effects

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and the relationship with the film microstructure, mechanical and barrier properties should be further investigated.

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2. Materials and methods

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2.1. Materials

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Chitosan, derived from crab shells (degree of deacetylation 85-90%, molecular weight

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80-120 KDa), was obtained from Sinopharm Chemical Reagent Co., Ltd. (Shanghai,

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China). Chilled meat was purchased from a local supermarket. All other reagents used were analytical grade. Deionized water was used in all the experiments.

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2.2. Preparation of films

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Chitosan powder (1%, w/v) was weighed accurately, and added into 0.5% (w/v) acetic acid solution. After stirring for 1 hour at room temperature, 10% glycerol (based on the mass of chitosan) was added to the film-forming solution as plasticizer. A 25 mL

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of film-forming solution was added into the square plastic Petri dishes (10 × 10 cm) and dried in an oven to a constant weight at 45, 55, 65, 75, 85 °C, respectively. The chitosan films were peeled off and maintained at 25 ± 1 °C and 53 ± 1% RH for 24 h. They were then soaked in 5% (w/w) sodium hydroxide (NaOH) solution for 50 s and 6

rinsed with deionized water several times to remove the excess alkali. They were finally dried at the corresponding temperatures for 30 min to obtain transparent chitosan films and conditioned at 25 ± 1°C and 53 ± 1% RH for more than 72 h prior to all the tests.

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2.3. Microstructure of films.

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2.3.1. Scanning electron microscopy (SEM)

Film strips (1 × 2 cm) were cryo-fractured by immersion into liquid nitrogen and conditioned in a desiccator at 25 °C before measurement. Cross-sections were cut

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from the conditioned samples and mounted on the specimen holder using

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double-sided adhesive tapes, and then sputter coated with gold under vacuum.

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Cross-section morphology of films was characterized by a field emission scanning electron microscope (SEM, S-4800, Hitachi, Japan) at an accelerating voltage of 5 kV.

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2.3.2. Fourier transform infrared spectroscopy (FTIR)

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FTIR spectra of films were recorded by attenuated total reflection (ATR) mode with a FTIR spectrometer (Nicolet IS 10, Thermo Electron, USA) at room temperature.

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Infrared spectra were recorded from 650 to 4000 cm-1 (64 consecutive scans with a 4 cm-1 resolution). Spectra were analyzed using the OMNIC 8.2 data collection

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software program (Thermo Fisher Scientific Inc., USA). 2.3.3. Small -angle X-ray scattering (SAXS) SAXS measurements were conducted on a Bruker NanoSTAR SAXS instrument (Bruker AXS, Germany). The micro-focus IμS-type generator operating at 50 KV and 7

600 mA were used at a Cu Kα radiation wavelength (λ) of 0.154 nm. The VANTEC-500 2D detector located at 1070 mm distance from the sample. The scattering data of the samples were recorded and the background scattering of the empty sample holder was subtracted after the absorption correction. The scattering

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curves were output from the plots of scattering intensity vs. the scattering vector, q =

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(4π/λ) sin θ (2θ = scattering angle). A generalized indirect Fourier transform program package was used to calculate the pair distance distribution function (PDDF). 2.3.4. X-ray diffraction (XRD)

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XRD patterns of chitosan films were obtained by an X-ray diffractometer (D2 Phaser,

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Bruker AXS Germany), and Copper target Cu Kα (λ = 0.15406 nm) and power 1600

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W (40 kV × 40 mA) were used. X-ray intensity was measured using a NaI crystal scintillation counter with a scanning range (2θ) of 5-60°, a step size of 0.04° and

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scanning speed was 6° min-1. The relative crystallinity degree of chitosan films was

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calculated using MDI Jade 6.5 software (Material date, Inc. Livemore, California,

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USA) according to (Lv et al., 2018). 2.4. Characterization of films. 2.4.1. Mechanical properties

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Mechanical properties were measured according to ASTM (2010) method, with slight modification. A texture analyzer (Stable Micro Systems, Surrey, UK) equipped with an A/MTG probe was used. The chitosan film was cut into 2 × 8 cm strips, and a micrometer (Dongguan, China) was used to determine the films thickness. The upper 8

and lower clamp distance was set at 50 mm, and the stretching speed and length was 0.5 mm/s and 10 mm, respectively. The curves of force versus the function of deformation was recorded with the texture Expert Exceed software (Version 2.64,

at break (%) were calculated using the following equations (Eq. 1 & 2): Maximum force (N) Thickness (mm) × width (mm) 𝐿 − 𝐿0 Elongation at break% = × 100 𝐿0 Tensile strength =

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Stable Micro Systems LTD. Godalming, UK). Tensile strength (MPa) and elongation

(1)

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(2)

where L0 represents the initial length of chitosan films and L represents the

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length of chitosan films at breaks. At least five repeats were performed for the final

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values.

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2.4.2. Water vapor permeability (WVP)

The water vapor permeability (WVP) and water vapor transmission rate (WVTR) of

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the chitosan films were measured, according to the method of ASTM (2004). A 10 mL

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of deionized water was added to the permeable cup, the chitosan film was then cut

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into a 4×4 cm piece and covered to the permeable cup and sealed it. After weighing the initial mass, the cup was stored in a desiccator with anhydrous silica gel for 72 h. The weight was monitored during storage. Each group had six parallel samples. The

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equation (4) and (5) given below as modified by Mchugh, Avena-Bustillos and Krochta (1993) were used to calculate the WVTR and WVP: Slope Film area WVTR × 𝐿 WVP = 𝑃𝐴1 − 𝑃𝐴2 WVTR =

(4) (5) 9

where PA1 is the partial vapor pressure at film outer surface, PA2 is the partial vapor pressure at film inner surface in the cabinet and L is the average thickness of chitosan films. 2.4.3. Oxygen permeability (OP)

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The OP was measured according to Chang, Liu, Sharif, Huang, Goff and Zhong

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(2019), with some modifications. A 5 mL of linoleic acid was added to a 30 × 50 mm

weighing bottle and covered with a piece of chitosan film (4 × 4 cm). Subsequently, the chitosan film was sealed at the mouth of the weighing bottle. After weighing the

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weight, the bottle was left at room temperature. The weight of the weighing bottle was

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monitored every 1 d for 5 d. The slope of each line was calculated by a linear

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regression equation (R2 > 0.99) of weight change vs. time. The oxygen permeability

(7): Slope Film area OPTR × 𝐿 OP = 𝑃𝐴1 − 𝑃𝐴2

(6)

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OPTR =

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transmission rate (OPTR) and OP were calculated by following the equation (6) and

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(7)

where PA1 is the air pressure at film outer surface in the cabinet, PA2 is the

oxygen partial pressure at film inner surface and L represents the average film

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thickness.

2.4.4. Water contact angle The contact angle of water on the chitosan film was measured using a contact angle meter by the drop method of Kraisit, Luangtana-Anan and Sarisuta (2015). After 4 μL 10

of deionized water was slowly dropped on the film surface with a 1 mL syringe (KDL Corp, Shanghai, China), the water contact angle was recorded immediately. the captured image was analyzed using the SCA20 software to calculate the angles. All the measurements were performed in triplicate.

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2.5. Chilled meat preservation experiment

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The chitosan films dried at 45, 65 and 85 °C were selected to carry out the chilled

meat preservation experiment. They were cut into 10 × 10 cm strips and wrapped around chilled meat pieces (15 g) in a sterile environment. The chilled meat without

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packaging was tested as blank sample, and the one wrapped with low density

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polyethylene (LDPE) film were used as the control sample. All the meat samples were

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stored in a refrigerator at 4 °C for 10 d. During the storage, the drip loss rate, thiobarbituric acid reactive substances (TBARS), aerobic plate count (APC), pH and

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2.5.1. Drip loss rate

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total volatile basic nitrogen (TVB-N) values of meat samples monitored.

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The drip loss rate of chilled meat was measured based on the method of Choe, Stuart and Kim (2016) The initial weight of the samples was weighed before packaging. At certain times, the chilled meat was taken out from the package and wiped with a tissue

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to remove the surface residual moisture. The meat was subsequently weighted. Three parallel samples were prepared for each sample. The drip loss rate was calculated according to the following formula (5): Drip loss (%) =

𝑊1 − 𝑊2 × 100 𝑊1

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where W1 represents the initial weight of the chilled meat and W2 is the final weight of the chilled meat. 2.5.2. Thiobarbituric acid reactive substances (TBARS) Analysis According to Rossi, Pastorelli, Cannata, Tavaniello, Maiorano and Corino (2013), 0.3

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g of chilled meat, 3 mL of 1% (w/w) 2-thiobarbituric acid (TBA) solution and 25 mL

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of 7.5% (w/w) trichloroacetic acid (TCA) solution were added into a test tube and

sealed with foil. After heating in a boiling water bath for 30 min, it was taken out and cooled to room temperature. A 4 mL of this suspension was mixed with 4 mL of

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chloroform and vortexed for 1 min. The resulting mixture was then centrifuged at

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4100g for 10 min. The absorbance was measured at 532 nm using a

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spectrophotometer with deionized water as the blank. The TBARS values of each group were measured in triplicate and calculated used the following formula (9): 𝐴532 × 9.48 𝑚

(9)

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TBARS value =

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where A532 is the absorbance at 532 nm of the mixture and m represents the

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actual weight of the chilled meat. The 9.48 is a constant based on the absorbance of a certain weight of thiobarbituric acid-malonaldehyde at 532 nm. 2.5.3. Aerobic plate count (APC) Analysis

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APC was determined according to the method of the International Standard (AFNOR). Accurately weighed chilled meat (9.0 g) was added into an aseptic sampling bag filled with 90 mL of normal saline. The sampling bag was beat for 2 min with a slap homogenizer. A 1 mL of the sample solution was serially diluted with a concentration 12

gradient and transferred to a Petri dish, followed by the addition of 15 mL of plate count agar medium at 46 °C. The dishes were shaken to ensure that the inoculation was uniformly mixed. The mixture was then cooled at room temperature until solidification. The petri dishes were inverted and incubated in an incubator (37 ± 1°C

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three times. Four replicates were performed for each sample.

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and 50 ± 1% RH). After 48 h of incubation, the APC value of per dish was recorded

2.5.4. pH analysis

After chopping the chilled meat in a sterile environment, 3.0 g of meat was added into

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an Erlenmeyer flask containing 27 mL of deionized water with vigorous stirring. The

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pH of the suspension was then measured using a pH meter (Mettler-Toledo,

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Switzerland). The measurements were performed in triplicate. 2.5.5. Total volatile basic nitrogen (TVB-N) Analysis

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The TVB-N content of chilled meat was measured using a semi-micro determination

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of nitrogen. A 5.0 g of chilled meat was added into a nitrification tube and thoroughly

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infiltrated with 27 mL of deionized water for 30 min. A Kjeldahl nitrogen analyzer was used to determine the TVB-N. The distillation time was set at 3 min and the titration endpoint was set as pH 4.65, and 30 mL of boric acid was used as the

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receiving solution. TVB-N was calculated according to the equation (10): TVB − N =

𝑉1 − 𝑉2 × c × 14 × 100 𝑚

(10)

where V1 is the volume of hydrochloric acid standard titration solution consumed by the chilled meat and V2 is the volume of hydrochloric acid standard titration 13

solution consumed by the control blank. c represents the concentration of the standard titration solution of hydrochloric acid and m is the weight of the chilled meat. 2.6. Statistical analysis Data was presented as mean value ± standard deviation of at least triplicates. SPSS

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24.0 package (IBM, New York) was used to analyze the data by one-way analysis of

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variance (ANOVA). Duncan's-multiple range test was applied to determine the significant differences of the mean values (P < 0.05). 3. Results and discussion

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3.1. Micromorphology of Chitosan Films

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After drying, homogeneous and transparent chitosan films with a slight yellow

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appearance were obtained at all temperatures. But the internal microstructure of films was significantly affected by drying temperature. As shown in Fig. 1, the

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cross-section of films dried at higher temperatures appeared more compact and

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smoother in comparison to those dried at lower temperatures. Films were mainly

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formed by the intermolecular hydrogen bonding between polymer chains during drying (Huber & Embuscado, 2009). Generally, lower drying temperatures resulted in lower drying rates. The chitosan polymer chains might show more entanglements due

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to more hydrogen bonding formed at lower drying rates. Slower drying might also allow for the rearrangement of polymer chains to form a more crystalline structure (Mayachiew & Devahastin, 2008). These together led to the rougher and looser micromorphology for the chitosan films dried at lower temperatures. 14

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Fig. 1. SEM micrographs (magnification 2400×) of cross section of chitosan films

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dried at different temperatures.

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3.2. Intermolecular Interactions within Chitosan Films

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The chemical interactions of chitosan films were investigated by FTIR in Fig. 2. All

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films showed a broad peak in the region of 3500-3000 cm-1, which assigned to the N-H stretching and O-H stretching vibrations of chitosan. The distinct amide I (C-O

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stretching) and amide II (N-H bending) band was observed at 1653 cm-1 and 1589

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cm-1, respectively (Homez-Jara, Daza, Aguirre, Munoz, Solanilla & Vaquiro, 2018). Besides, the peaks at 1153 cm-1, 1024 cm-1 and 893 cm-1 corresponded to C−O−C

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(bridge oxygen) stretching and C−O stretching, respectively (Rubilar, Cruz, Silva, Vicente, Khmelinskii & Vieira, 2013; Shahzadi et al., 2016; Wang, Dong, Men, Tong & Zhou, 2013). The position of these peaks did not significantly vary with drying temperature. But the transmittance of the peaks in the range of 1700-1500 cm-1

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decreased as drying temperature increased (Fig. 2b). This might be due to the decrease in intermolecular interactions (Kolhe & Kannan, 2003). Moreover, the N-H stretching region (3500-3000 cm-1) has been also used as a measure of intermolecular interaction (Kolhe & Kannan, 2003). The decrease in N-H stretching region further suggested the

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decrease in intermolecular interactions for chitosan films dried at higher temperatures.

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Fig. 2. FTIR spectra of chitosan films of chitosan films dried at different

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temperatures.

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3.3. Crystalline Structure of Chitosan Films 3.3.1. SAXS Analysis

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The changes in the overall size and dimension of micro-phases within chitosan films were investigated by SAXS, and the scattering intensity depended on the electron

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density difference that associated with nanoscale features (Corsello, Bolla, Anbinder, Serradell, Amalvy & Peruzzo, 2017; Liu, Cai, Li, Chen, Li & Li, 2016). As shown in Fig. 3a, the scattering intensity at low q value decreased with increasing film drying temperature. This decrease in scattering intensity suggested that the higher drying 16

temperature led to a decrease in electron density contrast between the crystalline region and amorphous region (Zhu, Li, Zhang, Li & Zhang, 2016). Fig. 3a also showed that the slope of scattering intensity decreased and the decay rate of scattering intensity slowed down at low q values. These indicated that the micro-region

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aggregation structure or the crystalline region became close to a spherical shape with

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a smaller size as drying temperature increased.

Furthermore, the Dmax (maximum dimension) and Rg (radius of gyration) values of micro-region aggregation were calculated according to the pair distance

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distribution function (PDDF). As shown in Fig. 3b, the decrease of Dmax in value from

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23.5 nm to 17.2 nm further verified the formation of a sphere-like shape when the

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drying temperature increased. The decreased Rg values from 7.1 nm to 5.3 nm (45 °C to 85 °C) also suggested that the high drying temperatures caused the reduction of the

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whole crystalline regions. These results were corresponding to the increased smooth

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and compactness of film cross-section as drying temperature increased (Fig. 1). The

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higher drying temperatures not only restricted the entanglements of chitosan chains because of the higher drying rates, but also promoted their mobility, and thus reducing the size of ordered regions and enlarging the amorphous region (Zhu, Li, Zhang, Li &

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Zhang, 2016).

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Fig. 3. (a) SAXS pectra and (b) corresponding Dmax (maximum dimension) and Rg (radius of gyration) values of chitosan films dried at different temperatures. (c) XRD

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patterns and (d) corresponding crystallinity degrees of chitosan films dried at different

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temperatures.

3.3.2. XRD Analysis

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The ordered crystalline structures of the chitosan films dried at different temperatures were also investigated by XRD (Fig. 3c). Chitosan is a semi-crystallinity polysaccharide, and its film generally has three crystal forms, like non-crystalline, hydrated crystalline and anhydrous crystalline, according to previous studies (Ogawa, Yui & Miya, 1992; Shahzadi et al., 2016). Herein, only two peaks at 11.4° and 22.8°, 18

respectively corresponding to the anhydrous crystalline and the typical fingerprint of chitosan film (Srinivasa, Ramesh, Kumar & Tharanathan, 2004), were observed. The absence of the peak at 8°, which assigned to the “Type II” polymorph of chitosan acetate (He, Ao, Gong & Zhang, 2011). This was due to the neutralization treatment

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for chitosan films. The dying temperature did not affect the position of these two

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diffraction peaks, suggesting that there were no changes in the inter-planar spacing of

associated crystallites (Zhu, Li, Zhang, Li & Zhang, 2016). However, the crystallinity degree of chitosan films gradually decreased from 26.3 % to 23.5% as drying

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temperature increased from 45 °C to 85 °C (Fig. 3d), which might be due to the

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reduction in ordered arrangement of chitosan molecules at higher drying temperatures.

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This decrease in crystallinity degree further confirmed the decrease in micro-region aggregation size and increase in film compactness when drying temperature increased

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(Fig.1 and 3a, b). According to above analyses, a schematic illustration was used to

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illustrate the decrease in intermolecular interactions between chitosan chains and the

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decrease in size of crystalline structures after drying at increased temperatures are

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shown in Scheme 1.

Scheme 1. Schematic representation of the microstructure changes within chitosan

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film with increasing drying temperature. 3.4. Mechanical Properties of Chitosan Films The mechanical properties of films are crucial for the application since they are associated with the durability and the ability to preserve the mechanical integrity of

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foods during handling, shipping, and storage (Shahbazi, 2017). Tensile strength

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represents the fracture resistance under tensile stress, and elongation at break accounts for the ability to resist shape changes before breaking (Homez-Jara, Daza, Aguirre, Munoz, Solanilla & Vaquiro, 2018). As shown in Table 1, drying temperature

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significantly affected the tensile strength of the chitosan films, where its value

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decreased from 129.2 ± 6.3 MPa at 45 °C to 109.9 ± 3.8 MPa at 85 °C (P < 0.05). The

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elongation at break also gradually decreased from 5.3% to 4.3% as drying temperature increased from 45 °C to 85 °C, but no significant differences were shown (P > 0.05).

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These suggested that higher drying temperatures resulted in less strong and less

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Maté (2010)

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flexible films, similar to those reported by Fernández-Pan, Ziani, Pedroza-Islas and

Faster drying rates at higher temperatures caused a much more serious structure

collapse of chitosan film with less ordered structures and weaker intermolecular

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interactions. But slower drying rates at lower temperatures allowed the chitosan chains to rearrange and to form a more ordered structure with stronger intermolecular interactions, as shown in Fig. 2 and 3. The ordered structures could also facilitate the sliding of the chitosan when stretching the film, where a more intense force was 20

required due to the higher number of inter-polymer hydrogens at the same time (Fernández-Pan, Ziani, Pedroza-Islas & Maté, 2010). Since there was no significant change in the thickness (P > 0.05) of chitosan films as drying temperature increased, these further suggested that the changes in mechanical properties were mainly due to

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the differences in the film structure.

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Table. 1. Thickness, tensile strength and elongation at break of chitosan films dried at different temperatures. Thickness (μm)

Tensile strength (MPa)

Elongation at break (EB%)

45 ℃

15.5±1.7a

129.2±6.3a

5.3±0.8a

55 ℃

18.0±0.8a

124.3±10.5ab

5.2±1.1a

65 ℃

15.6±1.7a

119.6±7.5b

4.9±1.7a

75 ℃

15.8±0.4a

115.5±3.3bc

5.0±1.3a

85 ℃

16.1±2.5a

109.9±3.8c

4.3±1.1a

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The type of film

*Values are mean ± standard deviation. Different letters in the table indicate

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significant differences (P<0.05).

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3.5. Water Vapor and Oxygen Barrier Properties of Chitosan Films The WVP and OP of films are two important features corresponding to their application as food packing materials (Dong, Yun, Li, Sun, Duan & Jin, 2015; Song,

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Song, Jo & Song, 2013). They respectively refers to the capacity of the film to hinder or to reduce the moisture or oxygen transfer between food and its external environments (Homez-Jara, Daza, Aguirre, Munoz, Solanilla & Vaquiro, 2018). The effect of drying temperature on the WVP of chitosan films are shown in Fig. 4a. The 21

WVP values ranged between 0.53 ± 0.01 g m-1h-1Pa-1 and 0.65 ± 0.03 g m-1h-1Pa-1 (P < 0.05), where the highest WVP value of 0.65 ± 0.03 g m-1h-1Pa-1 was observed for the film dried at 85 °C. However, opposite results were previously reported by Fernández-Pan, Ziani, Pedroza-Islas and Maté (2010) and Homez-Jara, Daza, Aguirre,

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Munoz, Solanilla and Vaquiro (2018), where the lowest WVP value was obtained at

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the highest drying temperature. They attributed this to the lower volume and thus

higher density within film matrix brought about by the more intense structural collapse drying at higher temperatures. In our study, the chitosan films were

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neutralized by the sodium hydroxide, whereas the previous reported films were dried

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directly without neutralization process. The hydrophilicity of chitosan materials could

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be reduced after neutralization, and the hydrophilicity of ordered structures within film matrix was highly reduced, according to Srinivasa, Ramesh, Kumar and

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Tharanathan (2004). These less hydrophilic ordered structures could increase the

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tortuosity of water diffusion through the film matrix (Ghanbarzadeh, Oleyaei &

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Almasi, 2015). Therefore, the ordered structure (crystal) content might mainly determine the moisture permeation when the chitosan film showed lower hydrophilicity, regardless of a more compact and dense film was obtained at higher

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drying temperature (Fig. 1). Similar results were also observed for OP values of chitosan films, where the OP

slightly increased in value (P > 0.05) as drying temperature increased from 45 °C (5.98 ± 0.35×10-5 cc/m·24h·atm) to 85 °C (6.62 ± 0.25×10-5 cc/m·24h·atm, Fig. 4b). 22

Chitosan films are generally good barriers for oxygen, which had similar oxygen barrier properties as commercially available polyvinylidene chloride or ethylene vinyl alcohol copolymer films, but superior to that of polyethylene films (Butler, Vergano, Testin, Bunn & Wiles, 1996). Moreover, Mcdonnell, Greeley, Kit and Keffer (2016)

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reported that the oxygen permeation through chitosan film was due to the association

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of O2 with NH3+. But the NH3+ in chitosan was transformed to NH2 during neutralization. Similarly, the tortuous path in films with more ordered structures could also restrict the oxygen permeation through chitosan film. Therefore, the OP value

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was also mainly controlled by the ordered structure content after drying.

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Fig. 4. (a) Water vapor permeability (WVP) and (b) oxygen permeability (OP) of

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chitosan films dried at different temperatures. 3.6. Surface Hydrophilicity/Hydrophobicity Properties of chitosan films For packaging film, the water contact angle is an direct indicator that reflects the

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hydrophilicity/hydrophobicity property (Han & Krochta, 1999). Generally, films with higher values of water contact angle have higher surface hydrophobicity (Tang & Jiang, 2007). The water contact angle value of < 65° indicates the hydrophilicity and of > 65° indicates the hydrophobicity of films, according to Ramos et al. (2013). As 24

shown in Table 2, the water contact angle values of chitosan films were all around 90°, and there were no significant differences between the drying temperatures (p > 0.05). The obtained chitosan films after neutralization were all hydrophobicity, and the surface hydrophilicity of films was independent of drying temperature. This further

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confirmed that the decrease in water barrier property of chitosan film as drying

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temperature increased was mainly caused by the decrease in the ordered structure content not the hydrophilicity/hydrophobicity properties (Fig. 4). Moreover, these

resultant chitosan films with higher hydrophobicity were appropriate for application

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in chilled meat preservation, since the meat contained a large amount of moisture

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within the tissue.

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Table. 2. Water contact angle of chitosan films dried at different temperatures. The type of film

55℃

85.3 ± 3.8a

65℃

90.2 ± 3.1a

75℃

87.1 ± 3.2a

85℃

91.0 ± 2.3a

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87.1 ± 6.0a

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45℃

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Water contact angle（°）

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*Values are mean ± standard deviation. Different letters in the table indicate significant differences (P<0.05).

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3.7. Preservation Effects of Chitosan Films on Chilled Meat

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3.7.1. Drip Loss Rate of Packaged Meat

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The capacity of juice retention is one of the major quality attributes for chilled meat, which determines the texture, health and consumer acceptability of meat (Cutter,

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2006). As a major problem in meat quality, insufficient juice retention capacity may

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cause the meat to be dark, hard and dry (Wu, Fu, Therkildsen, Li & Dai, 2015). Fig. 5a shows that the drip lose rate increased in value with storage day for all chilled meat

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samples. The chilled meat without packaging (blank) had the highest drip loss rate value during 10 d storage, which reached up to 45.6 ± 0.6% after storage. The blank sample also became more and more dark, hard and dry during storage (Fig. 5b). According to Shon and Chin (2008), the drip loss was mainly caused by the evaporation of water during storage. Thus, the water vapor barrier property of 26

package is the key to determine the performance in juice retention. However, the excessive low water vapor permeability of film might in turn speed up the spoilage of meat. As shown in Fig. 5, although the meat packaged with LDPE film showed the lowest drip loss rate value of 4.3 ± 0.3%, the meat sample was

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completely spoiled after 6 d storage without further monitoring. The meat packaged

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with chitosan films showed medium drip loss rate values between the blank and

LDPE sample. The drip loss rate value was negatively related to the film drying temperature, where the meat wrapped with chitosan film dried at 45 °C had the lowest

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drip loss rate value. This was due to the increase in the WVP value of chitosan film

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with increasing drying temperature (Fig. 4a). Kaewprachu, Osako, Benjakul and

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Rawdkuen (2015) had also reported similar results that the drip loss was related to the

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WVP values of films.

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Fig. 5. (a) Drip loss rate and (b) appearance of chilled meat packaged with chitosan films dried at different temperatures samples during 10 d storage at 4 °C. The chilled meat without packaging was tested as the blank sample, and the chilled meat packed by LDPE (low-density polyethylene) was tested as the control sample.

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3.7.2. TBARS of Packaged Meat

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Oxidative rancidity is one of the main factors that limit the shelf-life of chilled meat (Benjakul, Visessanguan, Phongkanpai & Tanaka, 2005). TBARS, corresponding to the secondary oxidation products, was measured to evaluate the oxidative stability of

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meat samples during storage and results are shown in Fig. 6a. The TBARS values of

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all meat samples increased gradually during storage, particularly for the blank sample

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without package. The meat packed with the LDPE film which had the highest increase. Moreover, there were no significant differences in TBARS values between the LDPE

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and blank sample (P > 0.05), which might be due to the negligible oxygen barrier

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properties of LDPE film (Gaikwad, Singh & Lee, 2017). The high oxygen content

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permeated through the LDPE film promoted the meat oxidation. The meat samples wrapped with chitosan films showed lower TBARS values, and lower film drying temperatures led to lower TBARS values. This might be attributed to the superior

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oxygen barrier properties of chitosan films, where the OP gradually decreased in value as drying temperature increased. Also, the antioxidant activity of chitosan could result in the TBARS value decrease by quenching the volatile aldehydes formed during oxidation such as malondialdehyde (Pabast, Shariatifar, Beikzadeh & Jahed, 28

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2018).

Fig. 6. (a) Thiobarbituric acid reactive substances (TBARS), (b) aerobic plate count

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(APC), (c) pH and (d) total volatile basic nitrogen (TVB-N) of chilled meat samples

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packaged with chitosan films dried at different temperatures during 10 d storage at 4 °C. The chilled meat without packaging was tested as the blank sample, and the chilled meat packed by LDPE (low-density polyethylene) was tested as the control

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sample.

3.7.3. APC of Packaged Meat Microbiological spoilage is another main factor that reduces the shelf-life of chilled meat during storage (Rao, Chander & Sharma, 2008). The spoilage microorganisms, 29

such as yeasts and molds, also lead to the presence of mucus and greening on the meat surface, which affect the sensory properties of chilled meat (Kaewprachu, Osako, Benjakul & Rawdkuen, 2015). As shown in Fig. 6b, the APC of meat samples all increased in value during storage. The LDPE film could highly slow down the

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microbial growth of meat as compared with the blank sample during 2 d storage.

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However, the APC value of meat wrapped by the LDPE film sharply increased to a higher value than that of the blank group after 4 d storage. This might be due to the high moisture environment around the meat caused by the strong water resistance

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capacity of LDPE film (Fig. 5), thus resulting in the rapid microorganism proliferation.

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The chitosan films effectively inhibited the microbial growth in chilled meat during

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whole storage time. But the drying temperature did not significantly affect the inhibitory effect, and all meat samples packaged with chitosan films had similar APC

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values. This indicated that the microbial growth inhibition of chitosan films on meat

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was mainly controlled by the antibacterial activity (Ma, Zhang, Critzer, Davidson,

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Zivanovic & Zhong, 2016; Pinto et al., 2012). 3.7.4. pH of Packaged Meat The pH value of fresh meat is generally below 6, and low pH values possess

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bacteriostasis function that regulate the microbial balance (Cullere et al., 2018). As shown in Fig. 6c, the pH value increased from 5.6 to the range of 6.4-6.9 after 10 d storage, which indicated that the chilled meat quality has been deteriorated. This increase in pH value was due to the production of alkaline autolysis compounds or 30

nitrogenous compounds and the formation of bacterial metabolites resulted from the protein degradation and microbial growth (Masniyom, Benjakul & Visessanguan, 2002; Pabast, Shariatifar, Beikzadeh & Jahed, 2018). The pH value of meat packed with the LDPE film also rapidly increased after 2 d storage, which was related to the

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large population of microorganisms (Fig.6b). The meat packed with the chitosan films

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had lower pH values than those of blank and LDPE sample after 10 d storage, which

displayed a negative correlation between the pH and the drying temperature. The lower pH values of meat samples wrapped with films dried at lower drying

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temperatures were mainly associated to the protective activity against substrate

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decomposition, not the microbial growth difference.

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3.7.5. TVB-N of Packaged Meat

TVB-N value, composing of ammonia and primary, secondary and tertiary amines, is

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another direct indicator that assesses the freshness of meat (Liu, Wang, Gui, Zheng,

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Dai & Li, 2012). The change of TVB-N value of chilled meat samples during storage

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is shown in Fig. 6d. At the first 2 d storage, the differences in TVB-N values between samples were not significant, indicating that the formation of amines was limited during the early storage time. Then, the TVB-N values were rapidly increased with

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storage time. This was due to the enzymatic degradation of spoilage bacteria or meat by the proliferation of microorganisms during storage (Cai, Chen, Wan & Zhao, 2011). The meat wrapped by LDPE film had the highest TVB-N value after 6 d storage, which was significantly higher than that of the blank sample. This was consistent with 31

the microbial growth of meat during storage (Fig. 6b). In addition to the LDPE sample, the blank sample showed the highest TVB-N value of 68.9 ± 2.3 mg/100 g after 10 d storage. The chitosan film samples showed much lower TVB-N values (36.4 – 47.2 mg/100 g) after 10 d storage. The differences in TVB-N values of chilled meat

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packaged with chitosan films dried at different temperatures became bigger with

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storage time, and higher drying temperatures led to higher TVB-N values. The inhibition effects of chitosan film against the generation of TVB-N had also been

reported by Jeon, Kamil and Shahidi (2002) in the preservation of cod and herring

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fillets.

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4. Conclusions

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In summary, a high drying temperature promoted the mobility but restricted the entanglements of chitosan chains within film-forming solution during drying, which

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resulted in decreased intermolecular interactions for resultant chitosan films.

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Micro-region aggregation structures with as smaller size and less ordered crystalline

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structures with a lower crystallinity degree were presented as drying temperature increased, leading to the compact and smooth internal microstructure of films. These changes in microstructure caused the decrease in mechanical and barrier properties of

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chitosan films mainly due to the reduced ordered structures at higher temperatures. But the surface hydrophilicity of films was independent of drying temperature. Moreover, chitosan films could maintain the quality and freshness of chilled meat for longer times than those of the LDPE film and blank sample. The preservation effects 32

of chitosan films on chilled meat decreased with increasing drying temperature, with higher values in drip loss rate, TBARS, APC, pH, and TVB-N. This study helps us in well understanding how drying temperature affects the microstructure and to associate with mechanical and barrier properties and preservation effects on meat, which could

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provide fundamental data for rationally design of chitosan-based film for food

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packaging.

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The authors declare no competing financial interest.

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Conflicts of interest

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Acknowledgements

This research was supported by National Key R&D Program of China

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(2016YFD0400802), National Natural Science Foundation of China (31801589),

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Natural Science Foundation of Jiangsu Province (BK20180615), China Postdoctoral

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Science Foundation (2017M621633). The research is also supported by national first-class discipline program of Food Science and Technology (JUFSTR20180204) and program of “Collaborative Innovation Center of Food Safety and Quality Control

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in Jiangsu Province”, China.

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