Development, physiochemical characterization and forming mechanism of Flammulina velutipes polysaccharide-based edible films

Accepted Manuscript Title: Development, physiochemical characterization and forming mechanism of Flammulina velutipes polysaccharide-based edible films Author: Hengjun Du Qiuhui Hu Wenjian Yang Fei Pei Benard Muinde Kimatu Ning Ma Yong Fang Chongjiang Cao Liyan Zhao PII: DOI: Reference:

S0144-8617(16)30824-4 http://dx.doi.org/doi:10.1016/j.carbpol.2016.07.035 CARP 11334

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

17-3-2016 30-6-2016 9-7-2016

Please cite this article as: Du, Hengjun., Hu, Qiuhui., Yang, Wenjian., Pei, Fei., Kimatu, Benard Muinde., Ma, Ning., Fang, Yong., Cao, Chongjiang., & Zhao, Liyan., Development, physiochemical characterization and forming mechanism of Flammulina velutipes polysaccharide-based edible films.Carbohydrate Polymers http://dx.doi.org/10.1016/j.carbpol.2016.07.035 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.

Development, physiochemical characterization and forming mechanism of Flammulina velutipes polysaccharide-based edible films Hengjun Du a, Qiuhui Hu a, Wenjian Yang a,*, Fei Pei a, Benard Muinde Kimatub,c, Ning Ma a, Yong Fang a, Chongjiang Caoa, Liyan Zhaob a

College of Food Science and Engineering/Collaborative Innovation Center for

Modern Grain Circulation and Safety/Key Laboratory of Grains and Oils Quality Control and Processing, Nanjing University of Finance and Economics, Nanjing 210023, China. b

College of Food Science and Technology, Nanjing Agricultural University, Nanjing

210095, China. c

Department of Dairy and Food Science and Technology, Egerton University, P.O.

Box 536-20115, Egerton, Kenya.

*

Corresponding author: Wenjian Yang

Phone/Fax: +86-25-86718519 E-mail address: [email protected].

Highlights 

Edible films were prepared with Flammulina velutipes polysaccharide (FVP) 1 



The FFS was a pseudoplastic fluid, and had appropriate viscosity for film-forming



The FVP films had desirable mechanical, barrier, and microstructure properties



The formation of FVP films depended on β-glycosidic and hydrogen bonds



FVP is a suitable material for edible film preparation 

ABSTRACT: Edible films of Flammulina velutipes polysaccharide were prepared and characterized in terms of rheological, optical, morphologic, mechanical and barrier properties to evaluate their potential application in food packaging. Results suggested that FVP film prepared by the solution of 1:150 (w/v) had the optimal mechanical property, smooth and uniform surface, and good barrier property to water (37.92±2.00 g mm/m2 h kPa) and oxygen (37.92±2.01 meq/kg). The capacity of film-formation might be related to inter-molecular and intra-molecular hydrogen bonds of FVP and formation of β-glycosidic bonds during the process of film-formation. These findings will contribute to a theoretical basis for the development of FVP film in food packaging.

Keywords: Edible film; Flammulina velutipes polysaccharides; Rheological property; Barrier property; Mechanical property; Microstructure.

2 

1. Introduction Edible films, as a kind of novel biodegradable natural materials, are mainly prepared from polysaccharides, proteins, and lipids. (Cerqueira et al., 2011; Galus & Kadzińska, 2015; Salarbashi et al., 2013; Valenzuela, Abugoch & Tapia, 2013). These films can be applied to the packaging of meat, seafood, vegetables, fruits, and candies for better barrier, antibacterial and antioxidant properties (Forato, de Britto, de Rizzo, Gastaldi & Assis, 2015; Tavassoli-Kafrani, Shekarchizadeh & Masoudpour-Behabadi, 2016; Volpe et al., 2015). Growing attention has been driven to search novel edible film materials from cereals, vegetables and fruits (Gutiérrez, Morales, Pérez, Tapia & Famá, 2015; Moreno et al., 2014; Salgado, Ortiz, Musso, Di Giorgio & Mauri, 2015). However, most of the films possess undesirable characteristics such as rough surface, poor mechanical or barrier properties. Therefore, it is further interest to seek new biomacromolecules with excellent film-forming abilities. Polysaccharides, mainly starch, cellulose, chitosan, alginate, and pullulan, are one of the most frequently used edible materials for development of edible film (Elsabee & Abdou, 2013; Gutiérrez, Morales, Pérez, Tapia & Famá, 2015; Wu et al., 2012; Xiao, Tong & Lim, 2012). However, these polysaccharides exhibit poor film-forming properties and by adding such additives, such as glycerol (Ghasemlou, Khodaiyan & Oromiehie, 2011), sorbitol (Talja, Helén, Roos & Jouppila, 2008), and polyethylene glycol (Cao, Yang & Fu, 2009), the film performance can be improved (Debeaufort, Quezada-Gallo & Voilley, 1998), which may bring bad flavor, taste into the film and ultimately affect edibility of films. Flammulina velutipes is a widely

3 

cultivated and commercially available mushroom in the world due to its desirable taste

and

high

nutritional

components

including

polysaccharides,

fungal

immunomodulatory proteins, and flavonoids. Our previous studies have proved bioactivity of F. velutipes polysaccharides (FVP) such as anti-proliferation activity and learning and memory improvement in rats (Yang, Fang, Liang & Hu, 2011; Yang et al., 2012; Yang et al., 2015). Another interesting finding is that the FVP showed excellent film-forming during the preparation process, which indicated the potential of FVP as a material of edible film. Moreover, no use of additives for the preparation of edible films makes FVP more attractive in terms of edibility safety. Up to today, no studies are available on the application of FVP in edible films. In this study, FVP was prepared and the rheological properties of film-forming solutions (FFS) were studied. Subsequently, edible films were prepared from FFS and the physical, mechanical and microstructure properties of films were investigated. Additionally, the mechanism for film-formation of FVP was analyzed using Fourier transform infrared spectroscopy.

2. Materials and methods 2.1 Preparation of FVP Fresh F. velutipes mushrooms were purchased from a local market (Nanjing, China). After washing, the mushrooms were dehydrated at an air temperature of 60 C for about 4.5h and a constant relative humidity of RH 20 % in an electric thermostatic drying oven (DNF610, YAMATO Scientific Co. Ltd, Japan). Dried F. velutipes was

4 

powdered and sieved through a No.100 mesh. A 200 g powder was extracted by 10 L deionized water with stirring for 5 h at 80 C. The extraction was collected by centrifugation (5,000 rpm, 10 min, 4 C). The supernatant was pooled and concentrated into one-tenth of the original volume under reduced pressure. Then, FVP extract was deproteinized four times with Sevag reagent (chloroform: butanol, 4:1) before precipitating with 4-fold volume anhydrous ethanol at 4 C for 12 h. After centrifugation at 10,000 rpm for 20 min, the precipitate was dialyzed in distilled water which was renewed every 2 h for 24 h at 4 C, and lyophilized as FVP.

2.2 Film preparation Preliminary experiments (data not shown) had demonstrated that the solid-to-liquid ratio higher than 1:300 (w/v) was not suitable for the preparation of films and the ratio lower than 1:300 (w/v) was used in this study. FVP was dispersed in distilled water following the solid-to-liquid ratio of 1:100, 1:150, 1:200, 1:250, 1:300 (w/v), stirred for 24 h at room temperature to achieve complete hydration, and degased for 15 min as FFS (S1:100, S1:150, S1:200, S1:250, S1:300). In order to speed up the dissolution of FVP, the distilled water was heated to 60 C. Then a 10 mL of solution was poured into disposable petri dish and left to dry in a biological cabinet at 25 C and RH 64 % for 12 h. The layers of five kinds of films prepared at ratio of 1:100, 1:150, 1:200, 1:250 and 1:300 (F1:100, F1:150, F1:200, F1:250, F1:300) were obtained for further experiments. Three replicates were prepared based at each solid-to-liquid ratio.

5 

2.3 Rheological properties of FFS The rheological properties of FFS were measured by a rheometer (MCR302, Anton Paar, Austria) equipped with cone-and-plate geometry of CP50-1 (diameter: 50 mm, cone angle: 1°, gap between cone and plate: 0.105 mm) at 25 C. All samples were sheared continuously at the shear rate ranging from 0.1 to 500 s-1. Three replicates were performed for each sample. Flow behaviors of the solutions were measured as a function of shear rate. Power law model as an equation was employed to examine flow properties of FFS (Eq. 1).   Κ  γn

(1)

where σ is the shear stress (Pa), γ is the shear rate (s-1), Κ (Pa s) is consistency coefficient and n is flow behavior index. Κ and n were determined at increasing shear rate (0.1–500 s-1) and calculated by Eq.1. The apparent viscosity (η, Pa s) values of FFS were calculated according to Eq.2. η  Κ  γ n 1

(2)

2.4 Fourier transform infrared spectroscopy A Bruker Tensor27 (Ettlingen, Germany) equipped with an attenuated total reflectance (ATR) accessory was used to record Fourier transform infrared spectroscopy (FT-IR) of FFS, which could suggest the change of chemical structure of film during film formation. FFS was dripped on the accessory, and scans were done at intervals of 5 min during 2 h at 25 C and 64 % RH. The spectra in the range of 4000 to 700 cm-1 were rationed and signal averages were collected for 50 scans at a

6 

resolution of 4 cm-1.

2.5 Film thickness An electronic digital micrometer (Guilin Guanglu Measuring Instrument Co. Ltd., China) was used to determine film thickness to the nearest 0.0001 mm. Ten measurements were made at random positions on each testing sample, and the mean values were calculated to analyze water vapor permeability (WVP) and tensile strength (TS).

2.6 Barrier properties 2.6.1 Water vapor permeability measurements The WVP was measured according to ASTM method E96 (1996) (Rezvani, Schleining, Sümen & Taherian, 2013) with some modifications. Briefly, polystyrene jars (ID: 3cm, height: 5.5 cm) were filled with deionized water up to 1cm from the jar mouth. The containers were then covered with FVP films without pores or any defects and further sealed with paraffin wax. The jars were placed in a constant temperature and humidity biological cabinet at 25 C and 50 % RH. A fan was operated within the cabinet to remove permeating water vapor from the surface of containers. The jars were weighted after 2 h, ensuring that the equilibrium has been reached and then weighed at intervals of 1 h for 10 h. The weight changes of the jars were recorded and the relation between the weight and time was plotted. Water vapor transmission rate (WVTR) was obtained from the slope of each straight line divided by fill area (m2),

7 

which was calculated by linear regression (R2>0.99). WVP (g mm/m2 h kPa) was calculated by Eq.3.

WVP 

WVTR D S(R 1  R 2 )

(3)

where WVTR is the measured water vapor transmission rate (g h-1 m-2) through the film specimen; S is the saturation vapor pressure of water (Pa) at test temperature (25 C); R1 is the RH of test environment (50 %) and R2 is the RH in the jars (100 %) ; D is the film thickness (mm). Determinations were made in triplicate.

2.6.2 Oxygen barrier Oxygen barrier property of FVP film was measured indirectly according to the method reported by Kurt and Kahyaoglu (2014). Fresh corn oil (50 mL) was poured into a 100 mL jar, which was covered with different FVP films, sealed with paraffin wax, and stored at 60 C for 10 d. Besides, in the same storage condition, two jars were filled with the same volume corn oil. One of them was sealed with a rubber plug for no oxygen penetration test (NOPT), and the other was kept open for no barrier test (NBT). The peroxide value (PV) of the corn oil was determined by sodium thiosulfate titration method. Three replications of all tests were measured.

2.7 Mechanical properties Tensile strength (TS) and the elongation at break (EAB), two typical measurements of mechanical properties, were determined using a texture analyzer (TA.XT2i Texture Analyzer, Stable Micro Systems, Godalming, UK) and measured 8 

according to ASTM standard method D882-02. The film specimens were equilibrated for 3 days at 25 C and 55 % RH, and then cut into rectangular sections (10cm×3cm). Film strips were clamped with grips, and measurements were carried out with an initial grip separation of 50 mm and a constant speed of 1mm/s at 25 C until rupture. Measurements were replicated at least eight times for each type of film. TS (MPa) and EAB (%) were calculated by Eq.4 and Eq.5, respectively.

TS 

Fmax A

(4)

where Fmax (N) is the maximum load, A (m2) is the cross-sectional area of the strip.

EAB 

ΔL  100 L0

(5)

where L0 (cm) is the initial height of film strips; ΔL (cm) is the increased height before break of film strips.

2.8 Scanning electron microscopy Morphology of FVP film was evaluated by scanning electron microscopy (TM3000 Tabletop Microscope, HITACHI, Japan). The films were mounted onto aluminum specimen stubs using double-faced adhesive tape and coated with a gold layer by a sputter coater (BAL-TEC AG, Balzers, Liechtenstein). All samples were examined using an accelerating voltage of 15.0 kV, and magnifications of 4000 and 5000 were used.

9 

2.9 Statistical analysis The measured data were analyzed by SAS® system, Version9.0 (SAS Institute, Cary, NC) and Origin Pro 8.0. Least significant differences (LSD) multiple comparison tests were then performed with a 95 % confidence level.

3. Results and discussion 3.1 Rheological properties of FFS Fig.1 showed that the rheological curves of FFS solutions. The steady shear viscosity of all FFS specimens dropped as the shear rate increased, which was a typical character for pseudoplastic fluid (n<1). As predicted, with the rise of solid-to-liquid ratio, the steady shear viscosity of all FFS declined. At a shear rate of 100 s-1, FFS at the solid-to-liquid ratio of 1:100, 1:150, 1:200, 1:250 and 1:300 (w/v) (S1:100, S1:150, S1:200, S1:250, S1:300) gave the viscosities of 0.061±0.011, 0.037±0.014, 0.024±0.017, 0.019±0.006 and 0.016±0.007 Pa s, respectively. As the result of increasing shear rate, the shear stress increased and FVP molecules were wrapped together or deformed along the flow direction, which was an indication of shear-thinning phenomenon, and the similar results were reported in many other biological macromolecular liquid with pseudoplastic properties (Bao et al., 2016; Silva-Weiss, Bifani, Ihl, Sobral & Gómez-Guillén, 2013). From the values of K and n related to the rheology of the FFS solution (Table 1) and the results of power law model (Fig.2), it can be seen that solid-to-liquid ratio of FFS showed a significant impact on these values (p < 0.01). The reduction of K values

10 

was observed with the concomitant increase of n values as the ratio increased. S1:100 gave the lowest n value, indicating it was pseudoplastic, therefore exhibits shear thinning. It has been reported that the high viscosity or gel-type structures of FFS can cause the difficulty for the removal of air bubbles and the viscosity lower than 0.7 Pa was, therefore, advised for better preparation of film layer (Cuq, Aymard, Cuq & Guilbert, 1995; Nair, Jyothi, Sajeev & Misra, 2011). Thus the range of FFS’s viscosity in this study (0.019~0.068 Pa s) was suitable for forming films.

3.2 Barrier properties of films 3.2.1 Water vapor permeability The water vapor permeability (WVP) of films is one of the most important properties of edible films for food packaging applications since it has direct influence on the shelf life of food products. The lower WVP of film is, the better the barrier property. FVP edible films exhibited good barrier property and the order of WVP for films prepared at varying ratio was 1:300 > 1:250 > 1:100 > 1:200 > 1:150, and the ratio 1:150 achieved the highest water vapor barrier. The WVP of F1:150 was close to the edible films reported by Acevedo-Fani et al. (Acevedo-Fani, Salvia-Trujillo, Rojas-Graü & Martín-Belloso, 2015) and Pan et al. (Pan, Jiang, Chen & Jin, 2014). And the WVP level of 1:150 (w/v) in the present study was lower than the corn starch edible films reported by Mali et al. (Mali, Grossmann, García, Martino & Zaritzky, 2006).Within the range of these solid-to-liquid ratios, the content of FVP molecules in per unit volume of FFS will be a major determinant for the quality of intermolecular

11 

cross-linking reactions. Although F1:100 had the biggest FVP content, it didn’t give the lowest WVP value, which might be attributed to some aggregates of FVP that are not completely dissolved in menstruum in S1:100. In such way, the cross-linkage reaction was affected, which lead to the nonuniform and heterogeneous films.

3.2.2 Oxygen barrier properties Peroxide value (PV) is another essential barrier property of edible film (Janjarasskul & Krochta, 2010) as the oxidation of lipids or food ingredients causes food deterioration and reduce the quality. Compared with the control, jars covered with these FVP edible films showed significantly reduced PV of oil (P<0.05), indicating that FVP films provided good barriers to oxygen (Fig.4). Films prepared at varying ratio exhibited different impact on PV and the order of PV was F1:300 > F1:100 > F1:250 > F1:200 > F1:150, which was similar to the results of WVP. Again, F1:150 had the greatest barrier to oxygen. The oxygen permeability of FVP films is affected by many factors, such as relative humidity and morphology such as thickness (Kurt & Kahyaoglu, 2014). In combination with the results from SEM, the surface of F1:250 and F1:300 were rough with varying degrees of cracks, which might be a result from the decline of oxygen resistance. The thin films prepared by S1:300 and S1:250 showed lowest performance than thick films (F1:150 and F1:200). Taking together, the FVP films showed better oxygen barrier ability (37.92±2.01 meq/kg), compared with other polysaccharides edible films made from pullulane-chitosan films (91.82±4.59 meq/kg) (Wu, Zhong, Li,

12 

Shoemaker & Xia, 2013) and salep glucomannan films (91.20±1.06 meq/kg) (Kurt & Kahyaoglu, 2014).

3.3 Mechanical properties of films Mechanical properties of films were related to distribution and density of intermolecular and intramolecular interactions in the network formed in polymeric films (Leceta, Guerrero & de la Caba, 2013). Tensile strength (TS) and elongation at break (EAB) were able to display the mechanical properties of the films (Mali, Grossmann, García, Martino & Zaritzky, 2006; Pan, Jiang, Chen & Jin, 2014). TS was determined at the specimen break point under tensile stress as the capacity of resistance to rupture, and EAB is the protraction in the specimen length from its original length to the break point length, which is associated with the elasticity of a polymeric material (Silva & Dujovne, 2013). The stress-strain curves (Fig.5a) show the maximum load (Fmax) on the films and break time of samples, which are used to calculate TS and EAB. The effects of solid-to-liquid ratio on the films’ mechanical properties are shown in Fig.5b. The influence of solid-to-liquid ratio on TS and EAB were quite opposite. TS increased firstly and then declined with the increase of solid-to-liquid ratio, and the opposite tendency was observed for EAB. F1;150 showed highest TS but lowest EAB, followed by F1:100, F1:200, F1:250 and F1:300. The great tensile strength was possibly due to the lowered mobility resulting from the intermolecular hydrogen-bonding interaction (Tian, Xu, Yang & Guo, 2011). The relatively higher

13 

concentration of FVP in the film probably resulted in higher hydrogen and covalent bonds between FVP molecules, bringing about the mechanical structure of a more compact network with more tensile strength and less elasticity (Silva et al., 2016). These results indicated that F1:150 was more resistant to deformation of its tridimensional network. The results on the mechanical parameters of FVP film were in agreement with some other polysaccharide films (Carneiro-da-Cunha et al., 2009; de Moura et al., 2009), and the similar behaviors and variation tendency were reported by Wu et al. (Wu et al., 2012). Besides, the TS of FVP film is stronger than the chitosan film and HPMC/WPI film reported by Silva-Weiss et al. (Silva-Weiss, Bifani, Ihl, Sobral & Gómez-Guillén, 2013) and Rubilar et al. (Rubilar, Zuniga, Osorio & Pedreschi, 2015).

3.4 Morphology of films The microscopic structure is a principal index for FVP films, which reflects the surface morphology and internal organization structure of the films. SEM analysis revealed that the film surface at the ratio 1:150 was homogeneous without any visible pores (Fig. 6A~6E). The cross-section image (Fig. 6F~6J) showed F1:150 exhibited rather compact and smooth structure. These results were in agreement with the results of WVP, PV, TS and EAB. Some rough aggregates of FVP and uneven structure in the surface of F1:100 can be observed (Fig. 6A), which could have been caused by the aggregation of insoluble FVP in the nearly saturated solutions. This affected the flatness and homogeneous

14 

distribution of polysaccharides, which also led to the appearance of interspace along the aggregations. As a result, the uneven aggregation tampered with the crosslinking structure between FVP. This could explain with the poor performance in mechanical and barriers properties tests of F1:100. Meanwhile, the cross-section image of F1:100 showed the unstable internal structure with little chasms. Smooth and uniform surface microstructures were observed in the SEM images of F1:150 and F1:200 (Fig. 6B, 6C), and the transversal surfaces were cohesive and dense, reflecting the great capability of tensile resistance and gas barrier. Wu et al. (2009) reported that inter-molecular hydrogen bonds of polysaccharides was formed, which led to the generation of double helical conformations and strong three-dimensional network structure during the film-forming process. In this study, the dense structure of FVP films may be also due to the formation of network structure of FVP, which need our further investigation. The noticeable cracks were observed on the surface of F1:250 and F1:300. At solid-to-liquid ratio of FFS, the FVP molecules were not sufficient to generate a dense structure network, which may lead to a decrease in hydrogen bonding between FVP molecules.

3.5 FT-IR analysis Based on the above results (Subsection 3.2, 3.3 and 3.4), S1:150 was chosen for further analysis by FT-IR. The FT-IR spectra of the solution within the range of 4000~700 cm−1 are shown in Fig.7, which used to characterize the variation of chemical bonds over the formation of FVP edible films. With the progress of water

15 

evaporating and chemical bonds changing, the FT-IR spectra was significantly altered at 3325, 2920, 1643, 1025, 890 cm-1 and in the range of 1500~1200 cm-1. Stretching vibration absorption of free O-H groups generally occurred at 3650 ~ 3580 cm-1 with strong and sharp peaks without any other interference peaks, then it shifted to low wave length (3325 cm-1) where absorption peak became broader. This was caused by the formation of inter-molecular and intra-molecular hydrogen bonds with the bond force constants decreasing (Kurt & Kahyaoglu, 2014; Silva et al., 2016; Zhang et al., 2013). The peak height dropped when the film was formed owing to water evaporation. The peaks for C-H emerged at around 3000 cm-1, and the bands below 3000 cm-1 (2920 cm-1) were attributed to the symmetric and anti-symmetric stretching of C-H in -CH3 and -CH2 functional groups (Piermaria et al., 2011). Carbonyl (C=O) presented two peaks: an asymmetrical stretching peak at 1650 cm-1 and a weak symmetric stretching peak at 1412 cm-1 (Casu, Scovenna, Cifonelli & Perlin, 1978). The peak at 1643 cm−1 coincided with peak of C=O in the spectrum, and could be assigned to the bending mode of O-H in water molecules, which suggested the existence of water. With the evaporation process of water, its peak area declined but nevertheless seemed unable to be dislodged from the film completely (Piermaria et al., 2011; Wu et al., 2012). The wave number at 1640 and 1410 cm-1 were the characteristic bands of amide (N-H), which showed the trace protein in the FVP. It may also suggest the structural presence of proteoglycan (Ren et al., 2014; Staroszczyk, Sztuka, Wolska, Wojtasz-Pajak & Kolodziejska, 2014). The adsorption peaks at 1245 and 1366 cm-1 was attributed to C–C band stretching vibrations from a

16 

ketone sugar and C-H (O-CH2) flexural vibrations, respectively (Zhang et al., 2013).The strong absorption peaks at 1025 cm−1 corresponded to C-O-C asymmetric and symmetric stretching modes in the pyranose form of sugars (Qiao et al., 2010; Zhang et al., 2013). Two shoulder bands at 1155 and 890 cm-1 are feature peaks of β-glycosidic bonds (Fariña, Viñarta, Cattaneo & Figueroa, 2009; Kozarski et al., 2012).

4. Conclusions To summarize, polysaccharides from F. velutipes was used to prepare an edible film. The rheology of FFS suggested the potential of FVP for development of edible films, and the edible films proved excellent properties at the solid-to-liquid ratio of 1:150 (w/v) with the highest TS, lowest EAB values, and desirable barrier property to water vapor and oxygen. Another benefit of this film is no use of additives compared to other edible film. This study gives a first insight in the research of the film-forming property of FVP and the potential application value in food packaging. Furthermore, considering the commercial reality, production cost and the development of circular economy, the processing by-products of F. velutipes, such as roots and stalks of mushroom, deserve to be considered as the materials of FVP edible films in the practical application.

Acknowledgements

17 

This work is financially supported by the Natural Science Foundation of Jiangsu Province (No. BK20141009), the Special Fund for Agro-scientific Research in the Public Interest (No. 201303080) and the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).

References Acevedo-Fani, A., Salvia-Trujillo, L., Rojas-Graü, M. A., & Martín-Belloso, O. (2015). Edible films from essential-oil-loaded nanoemulsions: Physicochemical characterization and antimicrobial properties. Food Hydrocolloids, 47, 168-177. Bao, H., You, S., Cao, L., Zhou, R., Wang, Q., & Cui, S. W. (2016). Chemical and rheological properties of polysaccharides from fruit body of Auricularia auricular-judae. Food Hydrocolloids, 57, 30-37. Cao, N., Yang, X., & Fu, Y. (2009). Effects of various plasticizers on mechanical and water vapor barrier properties of gelatin films. Food Hydrocolloids, 23(3), 729-735. Carneiro-da-Cunha, M. G., Cerqueira, M. A., Souza, B. W. S., Souza, M. P., Teixeira, J. A., & Vicente, A. A. (2009). Physical properties of edible coatings and films made with a polysaccharide from Anacardium occidentale L. Journal of Food Engineering, 95(3), 379-385. Casu, B., Scovenna, G., Cifonelli, A. J., & Perlin, A. S. (1978). Infrared spectra of glycosaminoglycans in deuterium oxide and deuterium chloride solution: quantitative evaluation of uronic acid and acetamidodeoxyhexose moieties.

18 

Carbohydrate Research, 63, 13-27. Cerqueira, M. A., Bourbon, A. I., Pinheiro, A. C., Martins, J. T., Souza, B. W. S., Teixeira, J. A., & Vicente, A. A. (2011). Galactomannans use in the development of edible films/coatings for food applications. Trends in Food Science & Technology, 22(12), 662-671. Cuq, B., Aymard, C., Cuq, J.-L., & Guilbert, S. (1995). Edible packaging films based on fish myofibrillar proteins: formulation and functional properties. Journal of Food Science, 60(6), 1369-1374. de Moura, M. R., Aouada, F. A., Avena-Bustillos, R. J., McHugh, T. H., Krochta, J. M., & Mattoso, L. H. C. (2009). Improved barrier and mechanical properties of novel hydroxypropyl methylcellulose edible films with chitosan/tripolyphosphate nanoparticles. Journal of Food Engineering, 92(4), 448-453. Debeaufort, F., Quezada-Gallo, J.-A., & Voilley, A. (1998). Edible films and coatings: tomorrow's packagings: a review. Critical Reviews in Food Science, 38(4), 299-313. Elsabee, M. Z., & Abdou, E. S. (2013). Chitosan based edible films and coatings: a review. Materials science & engineering. C, Materials for biological applications, 33(4), 1819-1841. Fariña, J., Viñarta, S., Cattaneo, M., & Figueroa, L. (2009). Structural stability of Sclerotium rolfsii ATCC 201126 β‐glucan with fermentation time: a chemical, infrared spectroscopic and enzymatic approach. Journal of applied microbiology, 106(1), 221-232.

19 

Forato, L. A., de Britto, D., de Rizzo, J. S., Gastaldi, T. A., & Assis, O. B. G. (2015). Effect of cashew gum-carboxymethylcellulose edible coatings in extending the shelf-life of fresh and cut guavas. Food Packaging and Shelf Life, 5, 68-74. Galus, S., & Kadzińska, J. (2015). Food applications of emulsion-based edible films and coatings. Trends in Food Science & Technology, 45(2), 273-283. Ghasemlou, M., Khodaiyan, F., & Oromiehie, A. (2011). Physical, mechanical, barrier, and thermal properties of polyol-plasticized biodegradable edible film made from kefiran. Carbohydrate Polymers, 84(1), 477-483. Gutiérrez, T. J., Morales, N. J., Pérez, E., Tapia, M. S., & Famá, L. (2015). Physico-chemical properties of edible films derived from native and phosphated cush-cush yam and cassava starches. Food Packaging and Shelf Life, 3, 1-8. Janjarasskul, T., & Krochta, J. M. (2010). Edible packaging materials. Annual Review of Food Science and Technology, 1, 415-448. Kozarski, M., Klaus, A., Nikšić, M., Vrvić, M. M., Todorović, N., Jakovljević, D., & Van Griensven, L. J. L. D. (2012). Antioxidative activities and chemical characterization of polysaccharide extracts from the widely used mushrooms Ganoderma applanatum, Ganoderma lucidum, Lentinus edodes and Trametes versicolor. Journal of Food Composition and Analysis, 26(1-2), 144-153. Kurt, A., & Kahyaoglu, T. (2014). Characterization of a new biodegradable edible film made from salep glucomannan. Carbohydrate Polymers, 104, 50-58. Leceta, I., Guerrero, P., & de la Caba, K. (2013). Functional properties of chitosan-based films. Carbohydrate Polymers, 93(1), 339-346.

20 

Mali, S., Grossmann, M. V. E., García, M. A., Martino, M. N., & Zaritzky, N. E. (2006). Effects of controlled storage on thermal, mechanical and barrier properties of plasticized films from different starch sources. Journal of Food Engineering, 75(4), 453-460. Moreno, O., Pastor, C., Muller, J., Atarés, L., González, C., & Chiralt, A. (2014). Physical and bioactive properties of corn starch – Buttermilk edible films. Journal of Food Engineering, 141, 27-36. Nair, S. B., Jyothi, A. N., Sajeev, M. S., & Misra, R. (2011). Rheological, mechanical and moisture sorption characteristics of cassava starch‐konjac glucomannan blend films. Starch‐Stärke, 63(11), 728-739. Pan, H., Jiang, B., Chen, J., & Jin, Z. (2014). Blend-modification of soy protein/lauric acid edible films using polysaccharides. Food chemistry, 151, 1-6. Piermaria, J., Bosch, A., Pinotti, A., Yantorno, O., Garcia, M. A., & Abraham, A. G. (2011). Kefiran films plasticized with sugars and polyols: water vapor barrier and mechanical properties in relation to their microstructure analyzed by ATR/FT-IR spectroscopy. Food Hydrocolloids, 25(5), 1261-1269. Qiao, D., Liu, J., Ke, C., Sun, Y., Ye, H., & Zeng, X. (2010). Structural characterization of polysaccharides from Hyriopsis cumingii. Carbohydrate Polymers, 82(4), 1184-1190. Ren, L., Hemar, Y., Perera, C. O., Lewis, G., Krissansen, G. W., & Buchanan, P. K. (2014). Antibacterial and antioxidant activities of aqueous extracts of eight edible mushrooms. Bioactive Carbohydrates and Dietary Fibre, 3(2), 41-51.

21 

Rezvani, E., Schleining, G., Sümen, G., & Taherian, A. R. (2013). Assessment of physical and mechanical properties of sodium caseinate and stearic acid based film-forming emulsions and edible films. Journal of Food Engineering, 116(2), 598-605. Rubilar, J. F., Zuniga, R. N., Osorio, F., & Pedreschi, F. (2015). Physical properties of emulsion-based

hydroxypropyl

methylcellulose/whey

protein

isolate

(HPMC/WPI) edible films. Carbohydrate Polymers, 123, 27-38. Salarbashi, D., Tajik, S., Ghasemlou, M., Shojaee-Aliabadi, S., Noghabi, M. S., & Khaksar, R. (2013). Characterization of soluble soybean polysaccharide film incorporated essential oil intended for food packaging. Carbohydrate Polymers, 98(1), 1127-1136. Salgado, P. R., Ortiz, C. M., Musso, Y. S., Di Giorgio, L., & Mauri, A. N. (2015). Edible films and coatings containing bioactives. Current Opinion in Food Science, 5, 86-92. Silva-Weiss, A., Bifani, V., Ihl, M., Sobral, P. J. A., & Gómez-Guillén, M. C. (2013). Structural properties of films and rheology of film-forming solutions based on chitosan and chitosan-starch blend enriched with murta leaf extract. Food Hydrocolloids, 31(2), 458-466. Silva, F., & Dujovne, D. (2013). Predictive mobility applied to content centric networks. Communications (LATINCOM), 2013 IEEE Latin-America Conference on (pp. 1-6): IEEE. Silva, F. E., Batista, K. A., Di-Medeiros, M. C., Silva, C. N., Moreira, B. R., &

22 

Fernandes, K. F. (2016). A stimuli-responsive and bioactive film based on blended polyvinyl alcohol and cashew gum polysaccharide. Materials science & engineering. C, Materials for biological applications, 58, 927-934. Staroszczyk, H., Sztuka, K., Wolska, J., Wojtasz-Pajak, A., & Kolodziejska, I. (2014). Interactions of fish gelatin and chitosan in uncrosslinked and crosslinked with EDC films: FT-IR study. Spectrochim Acta A Mol Biomol Spectrosc, 117, 707-712. Talja, R. A., Helén, H., Roos, Y. H., & Jouppila, K. (2008). Effect of type and content of binary polyol mixtures on physical and mechanical properties of starch-based edible films. Carbohydrate Polymers, 71(2), 269-276. Tavassoli-Kafrani, E., Shekarchizadeh, H., & Masoudpour-Behabadi, M. (2016). Development of edible films and coatings from alginates and carrageenans. Carbohydr Polym, 137, 360-374. Tian, H., Xu, G., Yang, B., & Guo, G. (2011). Microstructure and mechanical properties of soy protein/agar blend films: Effect of composition and processing methods. Journal of Food Engineering, 107(1), 21-26. Valenzuela, C., Abugoch, L., & Tapia, C. (2013). Quinoa protein–chitosan–sunflower oil edible film: Mechanical, barrier and structural properties. LWT-Food Science and Technology, 50(2), 531-537. Volpe, M. G., Siano, F., Paolucci, M., Sacco, A., Sorrentino, A., Malinconico, M., & Varricchio, E. (2015). Active edible coating effectiveness in shelf-life enhancement of trout (Oncorhynchusmykiss) fillets. LWT - Food Science and

23 

Technology, 60(1), 615-622. Wu, C., Peng, S., Wen, C., Wang, X., Fan, L., Deng, R., & Pang, J. (2012). Structural characterization and properties of konjac glucomannan/curdlan blend films. Carbohydrate Polymers, 89(2), 497-503. Wu, J., Zhong, F., Li, Y., Shoemaker, C. F., & Xia, W. (2013). Preparation and characterization of pullulan–chitosan and pullulan–carboxymethyl chitosan blended films. Food Hydrocolloids, 30(1), 82-91. Wu, Y., Geng, F., Chang, P. R., Yu, J., & Ma, X. (2009). Effect of agar on the microstructure and performance of potato starch film. Carbohydrate Polymers, 76(2), 299-304. Xiao, Q., Tong, Q., & Lim, L.-T. (2012). Pullulan-sodium alginate based edible films: Rheological properties of film forming solutions. Carbohydrate Polymers, 87(2), 1689-1695. Yang, W., Fang, Y., Liang, J., & Hu, Q. (2011). Optimization of ultrasonic extraction of

Flammulina

velutipes

polysaccharides

and

evaluation

of

its

acetylcholinesterase inhibitory activity. Food Research International, 44(5), 1269-1275. Yang, W., Pei, F., Shi, Y., Zhao, L., Fang, Y., & Hu, Q. (2012). Purification, characterization and anti-proliferation activity of polysaccharides from Flammulina velutipes. Carbohydrate Polymers, 88(2), 474-480. Yang, W., Yu, J., Zhao, L., Ma, N., Fang, Y., Pei, F., Mariga, A. M., & Hu, Q. (2015). Polysaccharides from Flammulina velutipes improve scopolamine-induced

24 

impairment of learning and memory of rats. Journal of Functional Foods, 18, 411-422. Zhang, Z., Lv, G., He, W., Shi, L., Pan, H., & Fan, L. (2013). Effects of extraction methods on the antioxidant activities of polysaccharides obtained from Flammulina velutipes. Carbohydrate Polymers, 98(2), 1524-1531.

25 

Figure Captions Fig.1

Characterization of steady shear of FVP solutions at 1:100 (A), 1:150 (B),

1:200 (C), 1:250 (D), 1:300 (E) solid-liquid ratio at 25 C. Viscosity and shear stress as functions of shear rate for all solutions. With the increase of shear rate, the viscosity decreased and shear stress rose in all FFS. Fig.2

Curves of power law model ( σ  Κ  γ n ) for FFS in different solid-liquid

ratio. Fig.3

The WVP of FVP edible films at varying solid-liquid ratio.

Fig.4

The PV of FVP edible films at varying solid-liquid ratio.

Fig.5 (a) The stress-strain curves of FVP edible films in different solid-liquid ratio. (b)Tensile strength (TS) and elongation at break (EAB) of FVP edible films in different solid-liquid ratio. Fig.6

SEM of morphology of FVP films. A to E showed the surface of the films

prepared at varying solid-to-liquid ratio from 1:100 to 1:300 (× 4k); F to J showed the cross section images of the films prepared at varying solid-to-liquid ratio from 1:100 to 1:300 (× 5k). Fig.7

FT-IR spectra of the FFS during film formation.

26 

Figure 1

0.01

A 1E-3 0.1

1

10 Shear rate (1/s)

100

1 0.1

1

1E-3 0.1

B 1

10 Shear rate (1/s)

100

Viscosity Shear stress 10

10 1 0.1

C

1

100

1E-3 0.1

D 1

10 Shear rate (1/s)

Viscosity Shear stress 10

10 1 0.1

1

0.01

1E-3 0.1

E 1

10 Shear rate (1/s)

27 

100

Shear stress (Pa)

10 Shear rate (1/s)

Viscosity (Pa s)

1

0.1

0.1

 

0.01

0.01

1E-3 0.1

1

0.01

Viscosity Shear stress 10

10 Viscosity (Pa s)

0.1

0.1

Shear stress (Pa)

1

1

0.1

100

Shear stress (Pa)

0.1

Shear stress (Pa) Viscosity (Pa s)

Viscosity (Pa s)

1

Viscosity Shear stress 10

10

10 Shear stress (Pa) Viscosity (Pa s)

Viscosity Shear stress

10

0.1

 

 

Figure 2

12

1:100 1:150 1:200 1:250 1:300

Shear Stress (Pa)

10 8 6 4 2 0

0

100

200

300 -1

Shear rate (s )

28 

400

500

Figure 3

2

WVP (g· mm/m · h· KPa)

1.6

a

1.4 1.2 1.0

b c

c

d

0.8 0.6 0.4 0.2 0.0

1:100

1:150 1:200 1:250 Solid-liquid ratio

29 

1:300

 

Figure 4

120

a

PV(meq/kg)

100 80

b

b

c

60 40

d

d

20 0

e 1:100 1:150 1:200 1:250 1:300NOPT NBT

30 

Figure 5 F1:100 F1:150

200

F1:200

150

F1:300

50

50 0 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 time (s)

a b c

c 40

F1:250

100

(b)

30 20 10

31 

c

1:100

b

a

TS EAB 40 d

30

a

20 10

d 1:150 1:200 1:250 solid-liquid ratio

1:300

0

EAB(%)

(a)

TS(MPa)

Strength (N)

250

 

Figure 6

32 

1025

Figure 7

0.8 0.6 0.4 0.2 0.0 4000

3500

87min 85min 80min 75min 70min 65min 60min 0min 3000 2500 2000 1500 -1 Wavenumber (cm )

33 

890

1650 1412 1245

1.0

2920

Absorbance

1.2

3325

1.4

1155

1366

1.6

1000

 

Table1 Values of power law parameters (K, n) of FFS at 1:100 (w/v), 1:150 (w/v), 1:200(w/v), 1:250 (w/v), 1:300 (w/v) solid-to-liquid ratio,a and the apparent viscosity (η) at 100 s-1. sample

K

n

R2

η

1:100

3.397±0.028

0.152±0.002

0.9855

0.0684±0.0016

1:150

1.897±0.017

0.174±0.003

0.9842

0.0423±0.0018

1:200

1.114±0.032

0.197±0.002

0.9786

0.0276±0.0024

1:250

0.854±0.021

0.209±0.005

0.9749

0.0224±0.0028

1:300

0.655±0.013

0.232±0.001

0.9800

0.0190±0.0019

a

Values are means ± standard deviations.

 

34