Physical, antibacterial and antioxidant properties of chitosan films containing hardleaf oatchestnut starch and Litsea cubeba oil

Accepted Manuscript Physical, antibacterial and antioxidant properties of chitosan films containing hardleaf oatchestnut starch and Litsea cubeba oil

Kewang Zheng, Wei Li, Boqiao Fu, Meifang Fu, Qiaolin Ren, Fan Yang, Caiqin Qin PII: DOI: Reference:

S0141-8130(18)31999-8 doi:10.1016/j.ijbiomac.2018.06.126 BIOMAC 9966

To appear in:

International Journal of Biological Macromolecules

Received date: Revised date: Accepted date:

27 April 2018 19 June 2018 25 June 2018

Please cite this article as: Kewang Zheng, Wei Li, Boqiao Fu, Meifang Fu, Qiaolin Ren, Fan Yang, Caiqin Qin , Physical, antibacterial and antioxidant properties of chitosan films containing hardleaf oatchestnut starch and Litsea cubeba oil. Biomac (2018), doi:10.1016/ j.ijbiomac.2018.06.126

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ACCEPTED MANUSCRIPT Physical, antibacterial and antioxidant properties of chitosan films containing hardleaf oatchestnut starch and litsea cubeba oil Kewang Zhenga, Wei Lia*, Boqiao Fua, Meifang Fua, Qiaolin Renb, Fan Yangb, Caiqin Qina** School of Chemistry and Materials Science, Hubei Engineering University, Xiaogan 432000, China

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State Grid Corporation of China, Beijing 100031, China

*Correspondence to: Wei Li (E-mail: [email protected])

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** Correspondence to: Caiqin Qin (E-mail: qincq@ hbeu.edu.cn)

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Abstract: More and more attention was attached to food safety, it is necessary to endow food packaging films with good antibacterial and antioxidant properties Edible films based on chitosan (CH), hardleaf

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oatchestnut starch (HOS) and litsea cubeba oil (LEO) were prepared by solution casting. The properties and structures of the blend film with different proportion (xCH/yHOS) were evaluated. The CH-HOS

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films were firstly prepared by blending CH solution with HOS paste. The tensile strength (TS) and DPPH radical scavenging ability of CH-HOS films increased from 27.33 MPa to 33.54 MPa and 20.67% to

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52.34%, respectively, and water vapor permeability (WVP) decreased from 1.531 × 10-11 g m-1pa-1s-1 to 1.491 × 10-11 g m-1pa-1s-1, with the HOS content increased from the ratio of 1:0 to 1:1. Then, the LEO was

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added to 1CH-1HOS films. Tensile strength (TS), water vapor permeability, moisture absorption and total soluble matter (TSM) of the 1CH-1HOS film were remarkably decreased with 16%LEO. Meanwhile, the static contact angle and antimicrobial activity of 1CH-1HOS-16LEO film increased significantly. Hence,

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this blend film system has great potential for food packaging in the future.

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Keywords: chitosan; litsea cubeba oil; packaging film; antioxidant activity; antimicrobial activity

1. Introduction

Recently, there has been an increasing interest in edible films made from degradable and natural polymers to take the place of synthetic polymers to avoid pollution.[1-5] Edible films could be an alternative to synthetic packaging materials due to their capabilities to prevent microbial growth, loss of aroma, moisture loss, solute transport, water absorption in the food matrix, or oxygen penetration.[6-9] Moreover, edible films exist independently, or acting as carriers of food additives, have been particularly considered in food preservation due to their ability to extend the shelf life.[10-12] Generally, the raw 1

ACCEPTED MANUSCRIPT materials of edible films are cheap, non-toxic, eco-friendly, many of them are considered as waste or byproducts, such as chitosan/chitin, starch, cellulose derivatives, gums, lipids, proteins and fish gelatin.[13, 14] Among various natural resources, chitosan and starch display specific benefits in packaging films.[15-19] However, the traditional food packaging film lacks the antibacterial effect, which makes the food become a nutritional hotbed for bacteria in the process of preservation, thus would cause damage to human health. On the basis of safety edible, CH-HOS-LEO films is prepared by doping HOS and -LEO to

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make it have good resistance to bacteria. Hardleaf oatchestnut is widely grow in the south of China, and

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its fruit is a traditional Chinese food with abundant starch, protein, dietary fiber, vitamins, various

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minerals, and excellent antioxidant activity used for beer brewing, tofu, noodles and feed processing.[20] At the same time, hardleaf oatchestnut fruit has been reported as a traditional Chinese herb to treat the disease like diarrhea, indigestion and relaxing congestion. However, according to the investigation, the

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abundant hardleaf oatchestnut fruit resource in China has not been used effectively. But, HOS film has poor physico-chemical properties, water resistance, and is difficult to process, which limits its

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applications. This constraint has led to the development of a way to improve starch-based film properties by blending them with several natural polysaccharides, such as chitosan.[21-25] Chitosan is a de-Nacetylated form of chitin, the second most abundant natural biopolymer obtained from the shells of crab,

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shrimp, etc.[26] And chitosan has attracted considerable attention as a potential food preservative because

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of its non-toxicity, biodegradability, bio-functions, biocompatibility, and antimicrobial activity.[27-29] But, chitosan and hardleaf oatchestnut starch offer poor barriers to moisture, and their antimicrobial

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activities are not good enough, which are the main obstacle in its application.[30] On the contrary, a variety of spices and herbs possess antimicrobial activity, as well as their extracts. Plant essential oils have been found to exhibit antibacterial and antioxidant properties,[31-34] and the plant essential oils in food systems

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have been reported in many literatures.[35-38] Incorporating essential oil into edible films has been shown to enhance antimicrobial activities and lower water vapor permeability.[39-42] Litsea cubeba oil (LEO) is

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extracted from litsea cubeba fruit and its main constituents[43] are citral (around 51%), D-limonene (18.82%) and linalool (2.36%); and the flash point, boiling point of it are around 70 oC and 230 oC, respectively. However, there are few reports on the effects of LEO on the physical and structural properties of chitosan/starch films. Therefore, the aim of this work was to develop an edible film based on CH, HOS, and LEO, and investigate the influences of the HOS to CH ratio and LEO content on the physical, antioxidant, and antimicrobial properties of the films. We anticipate the development of a novel edible film that combines the advantages of CH, HOS, and LEO. 2

ACCEPTED MANUSCRIPT Experimental CH (deacetylated degree: 90%, viscosity 68 cps at 25 oC) was purchased from Golden-shell Biochemical Co. Ltd, China. The hardleaf oatchestnut fruits from Jiangxi Province, China were ovendried at 50 oC for 24 h, and ground into flour with a pulverizer. The LEO (CAS: 68855-99-2) used in this study was provided by Jiangxi Sinco Medicine oil Co. Ltd, China. Glycerol, Tween 80, and acetic acid were analytical pure and purchased from Sinopharm Chemical Reagent Co. Ltd, China. All these raw

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materials were used as received without further purification.

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Film synthesis

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HOS was dissolved in distilled water at concentrations of 0.5, 2, and 8 g/100 ml, 20% (w/w, weight) glycerol was added and then heated to 90 oC and kept for 20 min to form a starch paste. CH solution (2

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g/100 ml) was prepared by dispersing 2 g of chitosan powder in 100 ml of acetic acid solution (1%, v/v) and 20% (w/w, weight) glycerol was added. A series of xCH-yHOS films was prepared by mixing 100 ml of CH solution with 100 ml of the HOS solutions (0.5, 2 and 8 g/100 ml). A series of 1CH-1HOS

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films incorporation LEO were prepared by mixing 100 ml of HOS solution (2 g/100 ml), 100 ml of CH solution (2 g/100 ml), 1% (w/w, total weight) Tween 80, and different concentrations of LEO (4, 8, 12,

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and 16% w/w, total weight) and then homogenized at room temperature at 8000 rpm using a homogenizer for 5 min. Then all the film-forming solutions were filtered with cheesecloth to remove

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insoluble constituents and were then vacuumed degasified at 35 oC with a vacuum pump for 2 h to remove air bubbles. The film forming solutions were casted in cubic Plexiglas moulds (110 mm × 110

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mm × 8 mm) and dried in a ventilated oven at 35 oC for 72 h. Finally, the films were peeled and conditioned at 25 oC, 50% relative humidity (RH) with a temperature and humidity chamber (Jinghong,

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China) for 5 days before testing. Samples nomenclature was xCH-yHOS or xCH-yHOS-zLEO, the x and y represent the mass ratio of CH and HOS in the blend film, and the z represents the LEO concentration

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in the blend film.

Mechanical properties

The mechanical properties of the films were determined according to the method described by Ojagh[30] with a Texture Analyzer (AG-IC, Shimadzu, Japan), operated with a 50 N load cell equipped with tensile grips (A/TG model). Films at 25 oC and 50% RH were cut into strips (4 mm × 75 mm) and measured. The initial grip separation and crosshead speed were set to 25 mm and 20 mm/min. The TS and percentage elongation at break (EAB) of the samples was calculated using the following equation:

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ACCEPTED MANUSCRIPT TS=

F dL

(1)

Where TS was the tensile strength (MPa), F was the maximum tension (N), d was the initial grip separation (25 mm), and L was the average film thickness (mm).

l1  l0  100% l0

(2)

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EAB=

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Where EBA was the percentage elongation at break (%), l1 was film elongation at the moment of

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rupture (mm), l0 was the initial elongation (25 mm). Water vapor permeability (WVP)

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The wvp of films were performed according to Peng et al.[44] with some modification. Films (70 mm diameter) were sealed onto permeation cups (height: 25 mm, inner diameter: 42 mm) filled with anhydrous calcium chloride. The permeation cups were covered with a film and applied beeswax to

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guarantee sealing. The permeation cups were then placed in a temperature and humidity chamber (Jinghong, China, 25 oC and 85% RH). Changes in weight of the cell were recorded to the nearest 0.001 g

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and plotted as a function of time. WVP was calculated as follows:

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WVP=

m L At P

(3)

Where △m was the weight of permeation cup gained by desiccant (g), L was the average film

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thickness (m), A was the permeation area (m2), △t was the time of permeation (s), and △P was the water

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vapor pressure difference across the film (Pa). Moisture absorption

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The films were cut into a square piece of 3.0 cm × 3.0 cm and dried at 35 oC, 30% RH for 36 h in a vacuum oven. The films were weighted to give the dried film mass (Wi). After weighing, they were placed in a temperature and humidity chamber (Jinghong, China) maintained at 85% RH and 25 ± 2 oC, to ascertain their obtain water absorption kinetics. The samples were removed from the desiccators after 48 h and weighed (W1). The moisture content (Mt) of the samples was calculated using the following equation:

（W1  Wi ) 100 Wi

Mt＝

4

(4)

ACCEPTED MANUSCRIPT Where Mt is the moisture content of the sample at a fixed time expressed on a dry mass basis (%); W1 is the mass of the film sample after exposure to 85% RH for 48 h, and Wi is the initial dry mass of the sample. Total soluble matter (TSM) The percentage of total soluble matter was determined according to the method adapted of Peng.et.al.[44] Samples (4.0 cm in diameter) were weighed (M0) and then directly immersed in 200 ml of

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distilled water and kept under orbital agitation (50 rpm) at 25 ± 5 oC. After 24 h immersion, the samples

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were removed from the solution and vacuum-dried at 105 oC for 24 h, and then they were re-weighed (M).

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The TSM was calculated as follows:

（M 0  M )  100 M0

(5)

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TSM=

Where M0 is the initial mass of dry matter (g) and M is the mass of insoluble dry matter (g).

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Contact angle measurements

Static contact angle of the films were measured in air using a contact angle analyzer (Attension,

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TL100, Finland) equipped with an Image Analysis Attachment. The film sample (2 cm2) was put on a

the film using a microsyringe.

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moveable horizontal sample stage; then, a drop of 20 μL of distilled water was placed on the surface of

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Determination of minimum inhibitory concentration (MIC) of LEO against bacterial strains MIC of LEO was determined according to the method of Wiegand, Hilpert, and Hancock.[45]

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Antimicrobial activity

Antibacterial properties of the films were determined by using the agar diffusion method on

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Escherichia coli and Staphylococcus aureus. Spread plates of BHI Agar were inoculated with 100 μL (107-109 CFU/mL) of these bacteria overnight grown. In the same way, different films were punched into discs of 6 mm diameter and then placed on the surface of the inoculated plate. Next, the plates were incubated at 37 oC for 24 h, and afterwards, the diameters of the inhibitory zone surrounding film discs were measured and used to evaluate the antimicrobial properties of these edible films. DPPH radical scavenging activity in vitro The reducing capacity of the films were measured according to the method [46] with minor modifications. Briefly, 2 g of different samples were dissolved in 50 mL water, and stirred slowly for 24 h 5

ACCEPTED MANUSCRIPT at room temperature to obtain an edible film soaking solution. Some 3.0 mL of different edible film soaking solution were mixed with 1.0 mL of DPPH methanol solution (1 mM). The mixture was shaken violently and kept at room temperature in dark for 30 min, and the absorbance at 517 nm was measured using a UV-Vis spectrophotometer (722, Jinghong, Shanghai, China). The activity was calculated using the following equation:

（As  A0 ) 100 AS

% DPPH Activity =

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(6)

Where As is the absorbance of the DPPH methanol solution and A0 is the absorbance for the sample

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extracts. Fourier transform infrared (FTIR) spectroscopy

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FTIR spectra of the films were obtained by using a FTIR model Vertex 70 Bruker spectrophotometer (Bruker, Germany) equipped with an attenuated total reflection (ATR) device (45o ZnSe) to investigate

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the probably interactions between CH, HOS and LEO. The measuring probe directly touched the surface of the films. All films were scanned from 600 cm-1 to 4000 cm-1 with a resolution of 4 cm-1 and 32 scans.

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Scanning electron microscopy (SEM)

The films were fractured in liquid nitrogen. Microstructure observations of the cross-section were

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observed using a JEOL JSM-6510 scanning electron microscope at 5 kV after gold sputter-coating.

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Statistical analysis

The experiments were replicated five times, the data were analyzed with the ANOVA procedure

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(SPSS software, Ver. 19) and differences among mean values were processed by the Duncan’s multiple range tests. Significance was defined at p < 0.05. A standard deviation (p-value < 0.05) at the 95% confidence level was used to compare all the parameters analyzed between control films and all other

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formulations.

Results and discussion

Mechanical properties Firstly, the mechanical properties of the films were given in Table 1, which included tensile strength (TS) and elongation at break (EAB). As shown in Table 1, the TS of the CH-HOS films increased with the amount of added HOS and reached a maximum value at 33.54 MPa when the HOS to CH ratio was 1:1, but decreased at higher HOS ratios. Similar results have been reported by Xu et al.[47] who found that the TS of the composite film reach to the maximum when the starch to CH ratio of 1:1. The increasing TS 6

ACCEPTED MANUSCRIPT of the films were attributable to the formation of inter-molecular hydrogen bonds between the OH- of the HOS and the NH3+ of the CH, and these was supported by the amino peak of CH film shifted from 1564 cm-1 to 1593 cm-1 of the CH-HOS film and the microstructure of CH-HOS film. And the decreasing TS of the films were probably due to HOS intra-molecular hydrogen bonds rather than inter-molecular hydrogen bonds, thus leading to a phase separation between HOS and CH. In general, starch films are very brittle, so the EAB of the films decreased with increasing HOS. The EAB decreased from 38.15% to

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34.67% as the HOS increased from a CH: HOS ratio of 1:0 to 1:1, nevertheless, it sharply decreased at

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higher HOS ratios. Due to the brittle of HOS film, the addition of too much starch lowered the flexibity of the film. Based on the data set obtained, it was suggested that the greatest integrity of the two polymers

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occurred at a CH to HOS ratio of 1:1.

When the CH to HOS ratio was 1:1, the mechanical properties of the films affected by LEO were

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also measured. The TS of the 1CH-1HOS films significantly decreased when LEO content increased from zero (35.34 MPa) to 16% (27.57 MPa). This behavior could be attributed to the partial replacement of

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stronger polymer-polymer interactions by weaker polymer-oil interactions in the film network, which may decrease the cohesion of the polymer network forces, thus reducing the TS. However, the EAB

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increased slightly when the LEO content increased from zero (42.17%) to 8% (45.24%), and sharply decreased thereafter. This phenomenon could be explained by the microstructure (Fig. 5). The effect of

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LEO incorporation observed in the present study was in agreement with Shen et al.[48], who found that adding 10% (w/w) citronella essential oil or 20% (w/w) cedarwood oil to CH films resulted in an considerable increased in the films’ EAB, but adding 20% (w/w) citronella essential oil or 30% (w/w)

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cedarwood oil to CH films resulted in a significantly decreased in the films’ EAB. When 8% LEO was incorporated, the structure of the film was not destroyed and the mobility of HOS and CH chains

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increased, thus the EAB improved slightly. Nevertheless, the oil droplets were gathered and embedded in the network and destroyed the continuous structure of the film when over 8% LEO was incorporated.

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Thus, for mechanical properties, the optimum concentration of LEO in 1CH-1HOS film was 8%. Water vapor permeability To determine the water vapor barrier property of the films, the water vapor permeability (WVP) examined. The WVP of the films at 85% RH and 25 °C are depicted in Table 1. CH film and HOS film had a WVP value of 1.531 × 10-11 g m-1pa-1s-1and 1.585 × 10-11 g m-1pa-1s-1, respectively: lower WVP values were obtained by mixing of CH and HOS. When the ratio was 1:1, the WVP was lowest of 1.491 × 10-11 g m-1pa-1s-1, this indicated that the structure of the films was the most dense. This result was similar to the trend in TS values. 7

ACCEPTED MANUSCRIPT When LEO was added to the 1CH-1HOS film, the water vapor barrier properties were improved; indicating that the incorporation of LEO into 1CH-1HOS film caused the films to become less water vapor permeable. The WVP of the films obviously decreased from 1.491 × 10-11 g m-1pa-1s-1 to 1.413 × 10-11 g m-1pa-1s-1 as the concentration of LEO increased from 0 to 8%, but decreased slightly from 1.413 × 10-11 g m-1pa-1s-1 to 1.383 × 10-11 at LEO contents over 8%. This behavior could be attributed to stronger hydrogen and covalent interactions between LEO and CH or HOS, which may have decreased the

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availability of hydrophobic groups to form hydrophilic bonds with water molecules to reduce the

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hydrophilicity of the film, and the oil phase induces an increase in the tortuosity for water transfer in the matrix, thus increasing the distance travelled by water molecules diffusing through the film,[49]

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subsequently decreasing the WVP. Nevertheless, the higher concentration (over 8%) of LEO destroyed the continuous structure of the film, thus the WVP decreased slightly.

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Moisture absorption

In the same way, the equilibrium moisture content (EMC) was considered a simple way to evaluate

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the hydrophilicity of edible films. The results of EMC of the films conditioned at 85%RH were shown in Table 1. The EMC of CH film, HOS film, and CH-HOS blend films are around 20%; because CH and

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HOS are hydrophilic materials, the combination of CH and HOS does not significantly improve the EMC of the films, however, the EMC of the films could be effectively reduced by adding hydrophobic

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compounds to polysaccharides films, and it decreased as the LEO content increased from zero (20.25%) to 16% (13.02%). This change could be explained by the formation of covalent bonds between the

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functional groups of LEO and CH or HOS chains, leading to a decrease in the availability of hydroxyl and amino groups, limiting polysaccharide-water interactions by hydrogen bonding, thus resulting in a

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decrease in the equilibrium moisture content composite films. Total soluble matter (TSM)

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Total soluble matter (TSM) was the soluble content of edible film in water that reflected the interactions among the different components. The TSM values of different films at 25 ± 5o C after 24 h dipping are listed in Table 2. The TSM of CH film and HOS film are 17.26% and 21.21%, respectively. When the HOS was added to CH, the TSM of the films was lower than that of the HOS films, and the lowest TSM value of 16.06% appeared at a CH to HOS ratio of 1:1. This data implied that some interaction occurred between CH and HOS. Here, the CH and HOS had relatively high molecular weights, so it was assumed that they were insoluble in water. On the other hand, the glycerol used as a plasticizer is soluble, hence a theoretical total soluble matter of 16.7%[39] was calculated (Table 2). As shown, the experimental TSM values for samples 8

ACCEPTED MANUSCRIPT containing LEO are lower than the theoretical value for complete loss of glycerol and much lower than that for complete loss of glycerol and LEO in water. Even though LEO is insoluble in water, if there was no interaction between LEO and CH or HOS, migration of the entire LEO component to the water phase is expected. Incorporation of LEO into 1CH-1HOS films at levels of 8% and 16% led to 20% and 34% reductions in solubility in water, respectively. This indicated that the presence of LEO could lead to some associations with CH or HOS.

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Contact angle

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The contact angle was the angle between the tangent line at the contact point and the horizontal line

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of the solid surface. The contact angles of the films were shown in Fig.1 and Table 1. Control CH film and HOS film displayed a very low contact angle for water of 74o and 78o showing its hydrophilic nature, which in agreement with the literature[50]. The plot showed that there was no obvious changed in the static

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contact angle with incorporating the HOS in CH film. However, the contact angle of 1CH-1HOS film was significant increased with the LEO addition. The result indicated that the LEO can notably improve the

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hydrophobic properties of chitosan blend films. It may be due to the formation of hydrophobic groups on the polymer surface.

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MIC of LEO

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The MIC is usually used to as a measure to evaluate the antibacterial performance of essential oils. The MIC values of LEO are shown in Table 3. The results showed that LEO had the inhibiting effect to E. coli and S. aureus, and the MIC of E. coli and S. aureus was about 600 ppm and 580 ppm, respectively.

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This was supported by Kim et al.[51] who found that the MIC value of citral on the E. coli (500 µL/mL), and supported by Onawunmi[52] who reported that the MIC values obtained for citral against S. aureus, E.

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coli, C. albicans and M. gypseum was 0.05% v/v. Antimicrobial activities

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The antimicrobial activities were exhibited by the inhibitory zone of the films incorporated with LEO against a Gram-negative bacterium, E. coli, and a Gram-positive bacterium, S. aureus. As shown in Table 4 and Fig. 2, the CH film, HOS film, and 1CH-1HOS film had weak antibacterial properties, because CH and HOS did not diffuse through the adjacent agar media in the agar diffusion test, only organisms in direct contact with the active sites of 1CH-1HOS were inhibited,[53] indicating that HOS had some antibacterial activities. However, positive inhibition was observed when LEO-containing films were tested. The mainly active components of LEO are citral and D-limonene.[43] Liu[54] reported that LEO shows significant antimicrobial activity to Vibrio parahaemolyticus, Listeria monocytogenes, and 9

ACCEPTED MANUSCRIPT Lactobacillus plantarum. As expected, the films’ antimicrobial activities were stronger at higher concentrations of added LEO. Nevertheless, the inhibitory effects did not reveal a significant increase upon increasing the level of LEO at its higher concentrations. This was probably attributed to the flocculation and coalescence that occurred at high concentrations of added LEO. In addition, the LEO was more effective against S. aureus bacteria than E. coli bacteria. This difference is related to the cell wall structures of bacteria so that E. coli bacteria are less sensitive to such agents. The result was agreed

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with results of Seyed and Masoud[55] for chitosan films incorporated with cinnamon essential oil.

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DPPH radical scavenging activity

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Generally, the antioxidant activity in vitro could be evaluated by a DPPH radical scavenging experiment. The DPPH radical scavenging activities of the edible film soaking solutions were examined. As shown in Figs 3 and 4, the CH film was observed a slight scavenging radical scavenging ability about

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7.78%, it can be attributed to the fact that free radicals can react with the residual free amino (NH2) groups to form stable macromolecule radicals, and the NH2 groups can form ammonium (NH3+) groups

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by absorbing a hydrogen from the solution.[56] However the HOS film showed excellent radical scavenging ability of about 88.45% may attribute to the reaction between the free radicals and the

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hydroxyl groups of HOS to form ammonium groups. When CH was incorporated with HOS, the DPPH radical scavenging activity of 1CH-1HOS film increased in over 8 folds in relation to CH film, indicating

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that the HOS could significantly improve the antioxidant activity of CH film. However, the DPPH radical scavenging activities were little increased upon incorporation of LEO, suggesting the incorporation of LEO could not effectively improve the antioxidant activity of 1CH-1HOS film, it may due to the aldehyde

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Structural properties

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group in LEO rather than the polyphenols compounds.

FTIR spectroscopy was used to investigate the possible molecular interactions between different components. The FTIR spectra of CH film, HOS film, 1CH-1HOS film, and 1CH-1HOS-8LEO film are

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shown in Fig. 5. In the spectra of CH film, the broad band located at 3324 cm-1 was caused by O-H stretching [30], and that at 1564 cm-1 corresponded to N-H blending (amide-II).[57] In the spectra of HOS film, the peak at 1640 cm-1 was due to the presence of bound water. The peaks appearing from 926 to 1080 cm-1 corresponded to C-O bond stretching. When different components are mixed, physical blends versus chemical interactions are indicated by changes in characteristic spectra peaks. In the spectrum of 1CH-1HOS film, the amino peak of CH shifted from 1564 cm-1 to 1593 cm-1, with the incorporation of HOS. The result showed that interactions occurred between the amino groups of CH and the hydroxyl groups of HOS. Upon the addition of 8% LEO, the 10

ACCEPTED MANUSCRIPT peak at 3337 cm-1 shifted to 3325 cm-1 and became flatter. This shift might have been induced by the formation of hydrogen bonds between the LEO and HOS or CH, which decreased the interaction between hydrophilic groups and water. Moreover, the sharp peak at 1026 cm-1 split into two shallower peaks (at 1027 cm-1 and 1048 cm-1), which might have been due to the interactions between the functional groups of LEO and HOS, or the formation of CH. All of these changes indicated that there were probably some interactions generated between LEO, HOS, and CH.[55]

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Microstructure

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The microstructures of the films were influenced by the arrangement of the different components in

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the matrix. Fig. 6 shows SEM micrographs of the cross-sections of CH film, HOS film, 1CH-4HOS film and 1CH-1HOS film containing various LEO concentrations. It should be noted that the CH film exhibited a smooth, compact, continuous structure, while the HOS film was rougher. It was noticed that the cross-

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sections of 1CH-1HOS film showed a compact surface without any grainy or porous structures, and without separation of phases between CH and HOS, indicating the good compatibility between CH and

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HOS. However, cracks were observed in the cross-sections of 1CH-4HOS film, suggesting that the higher content of HOS may cause the phase separation of HOS and CH in the composite film.

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The addition of LEO into 1CH-1HOS film led to a remarkable change in the cross-sectional topography, and a heterogeneous structure was generated. The microstructure of the film was slightly

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changed when 8% LEO was incorporated, but destroyed at LEO concentrations of over 8%. The LEO droplets were homogenously embedded in a continuous carbohydrate network, the number and the size thereof increased with increasing LEO concentration. This might have been due to the flocculation and

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coalescence occurring during drying. These results indicated that the presence of LEO disrupted interaction between HOS and CH in the film matrix. The reductions in TS and WVP of films with added

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Conclusion

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LEO can be explained by the changed film microstructures.

An edible film captured good antibacterial properties and antioxidant properties were successful prepared by solution casting based on CH, HOS and LEO. The results of this study showed that a unique compatibility can be achieved between appropriate proportion LEO, CH, and HOS. The TS, DPPH radical scavenging ability increased and WVP decreased, with the HOS content increased from the ratio of 1:0 to 1:1. The incorporation of LEO decreased the mechanical properties, water vapor permeability, moisture content, and total soluble matter of such films, but improved their antibacterial properties and static water contact angle. Structural characterization confirmed the good compatibility between CH and HOS and the interactions were present between the amino groups of CH and the hydroxyl groups of HOS. 11

ACCEPTED MANUSCRIPT The LEO droplets were homogeneously distributed throughout the films, and some interactions could occur between LEO and CH or HOS. The CH-HOS-LEO film based on CH, HOS, and LEO has good physical, antibacterial and antioxidant properties. This system based on edible films not only showed potential for use in food packaging, but also provide a reference for the further research in films of actual food environment.

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ACKNOWLEDGEMENTS

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This project was supported by the National Natural Science Foundation of China (Grant Number

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31371750) and the Educational Commission of Hubei Province of China (Grant Number Q20142703)

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ACCEPTED MANUSCRIPT Table 1 Effect of LEO incorporation on mechanical (TS and EAB), water vapor permeability (WVP), moisture content (EMC) and contact angle WVP g/pa·m·s）

Films

Thickness (mm)

TS (MPa)

EAB (%)

CH

0.120± 0.010b

27.33 ± 2.45a

38.15 ± 1.20a

1.531 ± 0.016a

19.74 ± 1.32a

1CH-0.25HOS

0.122± 0.008a

28.55 ± 2.36a

36.24 ± 1.33a

1.522 ± 0.015a

21.22 ± 0.79b

1CH-1HOS

0.127± 0.011b

33.54 ± 1.16b

34.67 ± 2.89b

1.491 ± 0.022b

20.25 ± 0.82b

1CH-4HOS

0.131± 0.007a

26.43 ± 1.36b

28.12 ± 4.66c

1.538 ± 0.017a

21.88 ± 1.35 a

HOS

0.133± 0.008a

26.55 ± 1.28b

20.33 ± 4.72c

1.585 ± 0.008c

23.75 ± 2.15c

78 ± 2.45b

1CH-1HOS-4LEO

0.128± 0.013c

32.25 ± 3.05c

35.65 ± 3.45d

1.467 ± 0.023b

18.21 ± 1.42a

83 ± 1.99b

1CH-1HOS-8LEO

0.129± 0.010b

30.63 ± 3.22c

37.24 ± 1.31a

1.413 ± 0.015a

16.24 ± 0.85b

88 ± 2.31b

1CH-1HOS-12LEO

0.131± 0.014c

28.07 ± 3.35c

30.28 ± 1.24a

1.395 ± 0.009c

14.85 ± 0.87b

91 ± 0.99a

1CH-1HOS-16LEO

0.134± 0.013c

27.57 ± 1.18b

24.58 ± 3.41d

1.383 ± 0.032d

13.02 ± 2.08c

94 ± 3.39c

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EMC (%)

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a, b, c, d

(10

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Different letters in the same column indicate significant differences among formulations (p< 0.05).

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Table 2 Effect of LEO incorporation on total soluble matter (TSM) Films

Experimental

-

-

16.06 ± 1.55

b

16.7

16.7

18.15 ± 3.58

d

-

-

21.21 ± 3.77

d

-

-

PT

14.62 ± 1.36b

16.1

19.4

1CH-1HOS-8LEO

12.21 ± 2.14c

15.6

21.9

c

15.2

24.2

b

14.7

26.5

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1CH-1HOS-4LEO

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1CH-1HOS-12LEO 1CH-1HOS-16LEO a, b, c, d

-

16.48 ± 0.97

1CH-1HOS

HOS

-

a

17.26 ± 1.06

1CH-0.25HOS

1CH-4HOS

Theoretical 2

ED

CH

Theoretical 1

a

11.25 ± 2.22 10.04 ± 1.38

Different letters in the same column indicate significant differences among formulations (p< 0.05). Values are mean ± standard deviation (n = 4) Theoretical 1: values were calculated assuming all the glycerol moves to the water phase. Theoretical 2: values were calculated assuming both, the glycerol and the LEO, move to the phase.

Table 3 MIC values of LEO against two bacterial strains. Bacteria

E.coli

S. aureus

MIC (ppm)

600

580

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Contact angle ( o) 74 ± 1.12a 77 ± 1.34a

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Fig. 1 Photos of the static contact angle of the films (a: CH film; b: HOS film; c: 1CH-1HOS film ; d: 1CH-1HOS-4LEO film; e: 1CH-1HOS-8LEO film; f: 1CH-1HOS-12LEO film; g: 1CH-1HOS-16LEO film)

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Table 4 Effect of LEO incorporation on antimicrobial activities Zone of inhibition (mm)

Films

E. coli Gram (-)

CH

7.55 ± 0.09a

6.25 ± 0.12a

1CH-1HOS

8.22 ± 0.12a

6.45 ± 0.14a

HOS

9.31 ± 0.26b

7.36 ± 0.24b

1CH-1HOS-4LEO

11.21 ± 0.13a

10.12 ± 0.29b

1CH-1HOS-8LEO

13.84 ± 0.25b

11.95 ± 0.44c

1CH-1HOS-12LEO

15.85 ± 0.48c

1CH-1HOS-16LEO

16.81 ± 0.67d

IP

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a, b, c, d

T

S. aureus Gram (+)

14.46 ± 0.62d 15.24 ± 0.67d

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Fig. 2 Photos of the inhibitory zone of the films (a: CH film; b: HOS film; c: 1CH-1HOS film; d: 1CH-1HOS-4LEO film; e: 1CH-1HOS-8LEO film; f: 1CH-1HOS-12LEO film; g: 1CH-1HOS-16LEO film)

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Fig. 3 DPPH radical scavenging activity of different films (a: DPPH methanol solution; b: HOS film; c:CH film; d: 1CH-1HOS film;

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e: 1CH-1HOS-8LEO film; f: 1CH-1HOS-16LEO film)

Fig. 4 Pictures of DPPH radical scavenging activity of different films

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Fig. 5 FTIR spectra of different films.

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(a: CH film; b: HOS film; c: 1CH-1HOS film; d: 1CH-1HOS-8LEO film)

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Fig. 6 SEM micrographs of cross-section of different films views at a magnification of 3000× (a: CH film; b: HOS film; c: 1CH-1HOS film; d: 1CH-4HOS film; e: 1CH-1HOS-8LEO film; f: 1CH-1HOS-12LEO film; g: 1CH-1HOS-16LEO film)

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ACCEPTED MANUSCRIPT Highlights

1. Hardleaf oatchestnut starch had excellent antioxidant activity, and could effectively improve the antioxidant activity of the films. 2. Litsea cubeba oil had excellent antimicrobial activity. 3. A unique compatibility can be achieved between appropriate proportion chitosan,

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hardleaf oatchestnut starch, and litsea cubeba oil.

4. The film prepared by chitosan, hardleaf oatchestnut starch, and litsea cubeba oil had good

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antimicrobial and antioxidant activity and physico-chemical properties.

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