Development and characterization of bacterial cellulose reinforced biocomposite films based on protein from buckwheat distiller’s dried grains

Accepted Manuscript Title: Development and characterization of bacterial cellulose reinforced biocomposite films based on protein from buckwheat distiller’s dried grains Author: Xuejiao Wang Niamat Ullah Xuchun Sun Yan Guo Lin Chen Zhixi Li Xianchao Feng PII: DOI: Reference:

S0141-8130(16)31313-7 http://dx.doi.org/doi:10.1016/j.ijbiomac.2016.11.106 BIOMAC 6799

To appear in:

International Journal of Biological Macromolecules

Received date: Revised date: Accepted date:

24-8-2016 23-11-2016 26-11-2016

Please cite this article as: Xuejiao Wang, Niamat Ullah, Xuchun Sun, Yan Guo, Lin Chen, Zhixi Li, Xianchao Feng, Development and characterization of bacterial cellulose reinforced biocomposite films based on protein from buckwheat distiller’s dried grains, International Journal of Biological Macromolecules http://dx.doi.org/10.1016/j.ijbiomac.2016.11.106 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 and characterization of bacterial cellulose reinforced biocomposite films based on protein from buckwheat distiller’s dried grains Xuejiao Wanga, Niamat Ullaha,b, Xuchun Suna, Yan Guoa, Lin Chena, Zhixi Lia, Xianchao Fenga,* a

College of Food Science and Engineering, Northwest A&F University, No. 22

Xinong Road, Yangling, Shaanxi 712100, China b

Department of Human Nutrition, The University of Agriculture Peshawar, Khyber

Pakhtunkhwa 25000, Pakistan

*Corresponding author: Xianchao Feng Associate Professor, College of Food Science and Engineering, Northwest A&F University, No. 28 Xinong Road, Yangling, Shaanxi, China 712100. Email address: [email protected]. Tel/Fax: 86029-87092486.

Graphical abstract

High lights 1. Bio-composite film was prepared with protein extracted from buckwheat DDGS. 2. BC is an excellent reinforcement for protein films with high purity. 3. Mechanical, barrier, color, DSC, FTIR, and viscosity properties were evaluated. 4. Addition of BC could promote the mechanical properties and water vapor permeability of the films. 5. There is good compatibility between BC and protein matrix.

Abstract Biocomposite films were manufactured by combining protein extracted from buckwheat distiller’s dried grains with bacterial cellulose (BC). The film microstructures showed that BC is compatible with protein matrix and endows the film with high rigidity. Differential scanning calorimetry (DSC) showed that BC can promote thermal stability of the composite films. BC promoted the transition from a

Newtonian to a non-Newtonian fluid and the shear thinning behavior of protein-BC solution. Fourier Transform Infrared (FTIR) spectroscopy showed the main functional groups’ absorption peaks shifted to lower wavenumbers. Results of both FTIR and viscosity analysis proved the formation of intermolecular interactions through hydrogen bonds. These bonds affected film characteristics such as moisture content (MC), water solubility (WS), and water vapor permeability (WVP), which decreased with addition of BC. The WVP (6.68 ± 0.78 ~ 5.95 ± 0.54×10-10 g m/Pa s m2) of the films were lower than other protein films. Tensile strength (TS) values of films containing 1.8% and 2.0% BC (14.98 ± 0.97 and 15.03 ± 2.04 MPa) were significantly higher than that of pure protein films (4.26 ± 0.66 MPa). Combination of proteins extracted from a waste product and BC led to composite films with low water vapor permeability and excellent mechanical properties. Key words: buckwheat distiller’s dried grains, bacterial cellulose, biocomposite films

1. Introduction Biodegradable films can be relatively inexpensive to produce if derived from byproducts of agriculture and promise to be useful as packaging materials that can help solve environmental problems caused by synthetic polymers [1-3]. Agricultural wastes contain ample amounts of biopolymers such as proteins, polysaccharides, lipids, or their composites that can be manufactured into product packaging [2, 4, 5]. There is a growing interest in using proteins from amaranth, rice bran, soy, peanut, cottonseed, whey protein and corn to make biodegradable films. However, protein films tend to have poor water resistance and mechanical properties that allow the products inside to be affected by the environment. Also, these films increase the risk of package breakage in transport, sale or use, resulting in limited packaging application [6]. Several approaches have been tried in order to overcome the disadvantages of protein-based films, for instance, blending proteins with other biodegradable polymers or lipids, coating, lamination, and plasticization, altering the pH, and cross-linking by heat, chemicals, enzymes or irradiation. Biodegradable polymers, such as zein nanoparticles and cellulose fibers, have been used as reinforcements to improve the moisture barrier, mechanical and other properties of protein-based films [7-11]. Recently, numerous studies have focused on the use of cellulose fiber as reinforcing fillers in polymeric matrices. Cellulose fibers present an environmental advantage and, in biocomposites, add high strength, high hardness and low density [12]. Fibers from hemp, cotton, wheat straw, sugarcane and firs along with the keratin

or lignin inherently present in such vegetative tissues have been tested in the preparation of composite films [10]. Nevertheless, purifying fibers from plant waste materials takes time, energy and money. On the other hand, bacterial cellulose (BC) offers higher purity and higher yields than production of vegetable fibers. Importantly, BC has a high water-holding capacity (WHC), a low water release rate (WRR), high crystallinity, high porosity, an excellent mechanical strength, an ultra-fine web-shaped fiber network, it is biocompatible and biodegradable, and can be molded into various forms during production [13, 14]. Recently, a few studies have investigated the effects of adding BC to films, resulting new biocomposite materials. A biocomposite film of gelatin and BC had improved moisture sorption, WVP behavior, thermal stability and dynamic mechanical properties [15]. Furthermore, chitosan films made with different amounts of BC present significantly better mechanical properties and have a reasonable thermal stability and a low O2 permeability[16]. BC was found to be a better reinforcing agent than other fiber sources and can be added to a protein matrix to improve water vapor permeability and mechanical properties of biocomposite films. Distiller’s dried grains (DDGS) is a byproduct of the brewing industry and a multiple-component biowaste solid containing proteins (26.8 - 33.7%, dry weight basis), carbohydrates (39.2 - 61.9%, including fibers), oils (3.5 - 12.8%), and ash (2.0 - 9.8%) [17]. The volume of DDGS is increasing rapidly all over the world, particularly in China and the United States in recent years, with most of the increase due to ethanol production [18-20]. Over 20 million tons of stillage are produced from the liquor industry every year in China, of which DDGS is a major portion [21].

Therefore, there is interest in uses for the DDGS generated during alcohol production. Currently there are limited uses of DDGS, such as to feed stock animals, which cannot consume all the available stillage. Consequently, using DDGS as a base material for production of biodegradable films could be a potential way to improve its utilization and expand its application. The proteins in DDGS can be extracted in a simple and environmentally safe way. Furthermore, protein extracted from sorghum DDGS had excellent film-forming properties [22]. Yet, to the best of our knowledge, research focused on using the protein from DDGS to make biopolymer films remains relatively unpublished. In the present study, a film produced using protein extracted from buckwheat distiller’s dried grains and BC into the protein matrix to overcome low tensile strength and poor moisture barrier properties of films prepared by. The aim of this research was to study the effect of BC addition on the water solubility, moisture content, water vapor permeability, color, light transmission, opacity, mechanical properties, microstructure, thermal properties, and chemical structure of a protein-based film. It is hoped that the results of this study will promote the re-use of distiller’s dried grains and reduce environmental pollution. 2. Materials and methods 2.1. Materials Buckwheat distiller's dried grains (DDGS) were supplied by Buckwheat Liquor Co. Ltd. (Ziyang, Shaanxi, China). All chemicals were purchased from Sigma–Aldrich Chemical Co. (St. Louis, MO, USA).

2.2. Preparation of protein solution Protein from buckwheat DDGS was extracted according to Yang, Lee, Won and Song [22], with some modification. Buckwheat DDGS were ground at 10,000 rpm using a blender (FW80, Taisite Instrument Co., Ltd., Tianjin, China). The ground buckwheat DDGS was mixed with 7 volumes of distilled water and pH of the suspension was adjusted to 8.0 using 1 M NaOH. The suspension was stirred for 3 h at 80 °C and then centrifuged at 10,000 x g 15 min. Ammonium sulfate (70%) was used to precipitate proteins, which were collected after centrifugation. Pellet was dialyzed using a 8000-14000 Da dialysis membrane (Solarbio Technology Co., Ltd., Beijing, China). Protein concentration was measured using the biuret method and adjusted to 5%. 2.3. Production of bacterial cellulose BC was produced with G. hansenii CGMCC3917 according to a published method from our Lab [14, 23]. After harvest, BC sheet were rinsed with running tap water overnight, followed by soaking in a solution of 0.1 mol/L NaOH at 80 °C for 2 h. In order to ensure complete removal of alkali, BC was washed with deionized water several times. The BC was ground for 2 min using a soybean milk maker (Joyoung, Hangzhou, China), and then homogenized 3 times at 20 MPa with a high pressure homogenizer (SRH60-70, Shenlu Co., Ltd., Shanghai, China). After centrifuging at 10 000 x g for 15 min, the moisture content of the BC was measured and recorded. 2.4. Film preparation

Ten milliliter of the DDGS protein solution (5%) was mixed with sorbitol (0.25 g, half of the weight of the protein). The wet BC from above was converted into dry BC according to the moisture content and added to the mixtures for final BC contents of 1.1, 1.6, 1.8 and 2.0% (based on protein weight). The mixed solution was then homogenized at 20,000 rpm for 1 min by a homogenizer (IKA, Ultra-Turrax T18, Staufan, Germany). The suspension was heated at 70 °C for 0.5 h, and degassed by slow stirring. At last, the suspension was poured onto a Teflon-coated plate and dried in an oven at 40 °C for 24 h. The films were then conditioned in a thermo-hygrostat at 25 °C and 50% relative humidity (RH) for 48 h. 2.5. Scanning electron microscopy (SEM) SEM was used to examine the surface and cross-section microstructure of protein and protein-BC films. The film samples were cut and held with an aluminum tape and then sputtered by gold in an E-1045 sputter coater (HITACHI, Tokyo, Japan). All specimens were examined with a S-4800 SEM (HITACHI, Tokyo, Japan) under high vacuum condition and at an accelerating voltage of 10.0 kV. 2.6. Fourier transform infrared (FTIR) spectroscopy FTIR spectra of dried films were carried out using a Bruker Vertex-70 (Germany). The spectra were recorded in the range of 4000–400 cm−1 with a resolution of 4 cm−1. 2.7. Differential scanning calorimetry (DSC) DSC analysis was performed using a differential scanning calorimetry (Q2000, TA, USA). The samples (1.5 mg) were hermetically sealed in aluminum pans and heated at the rate of 5 °C min-1 from 20 °C to 250 °C.

2.8. Viscosity measurements of the film-forming solution (FFS) Steady state flow measurements of the film-forming solution (FFS) were performed at 25 °C according to the method described by El Miri, Abdelouahdi, Barakat, Zahouily, Fihri, Solhy and El Achaby [24], using a rheometer (ZX7MAR1000, Tenn., U.S.A.). The steady shear flow was measured from 0.1–100 s−1 and flow curves of all FFS were obtained. 2.9. Film thickness The film thickness was determined using a digital micrometer with accuracy to 0.01 mm (Guanglu, 111N-101GA, Guilin, China). Ten different positions were measured and averaged. 2.10. Water solubility (WS) The WS of the films was measured according to the method described by Atef, Rezaei and Behrooz [25]. Briefly, samples were first cut to size (4 × 4 cm) in triplicate and dried at 105 °C for 24 h to determine initial dry weight. The dried films were then immersed in 50 ml of distilled water and stirred at 100 rpm for 24 h at room temperature. After 24 h, any remaining pieces of films were taken out and dried at 105 °C for 24 h to obtain the final dry weight. Water solubility (%) was calculated using the following equation: WS (%) = initial dry weight - final dry weight ×100 initial dry weight

2.11. Moisture content (MC) For measuring MC, films were uniformly cut to size (2 × 2 cm) in triplicate and dried in an oven at 105 °C for 24 h. After drying, the dry weight of the film was

recorded, and the MC was calculated using the following equation: MC (%) = initial sample weight - dry sample weight 100 initial sample weight

2.12. Water vapor permeability (WVP) The WVP was measured according to the method described by Jiang, Xiong, Newman and Rentfrow [26] with a minor modification. A polypropylene centrifuge tube of 50 ml (482.8 mm2 mouth area), containing 30 g of silica desiccant beads, was covered with the films (5×5 cm), and placed in a thermo-hygrostat (HengTai FengKe, HT/GDSJ, Beijing, China) at 20 °C with 80% RH. Weight was recorded for 8 h with 30 min interval, and the plot of weight gain versus time was obtained. The slope of the plot is water vapor transmission rate C (g / h). WVP of the films was recorded with following formula: WVP =

Cn A  P

Where, C is water vapor transmission rate (g / h), n is thickness of the film (mm), A is the effective area of the film (mm2), ∆P is water vapor pressure difference on both sides of the film (Pa). 2.13. Color, light transmission and opacity measurements A colorimeter (Minolta, CR-310, Tokyo, Japan) along with a Hunter L* a* b* color space was used for color assessment. Measurements were done in triplicates. The standard plate was L* = 97.43, a* = 0.41, b* = 1.84 and each film was placed on a white standard plate. The total color difference (∆E) was obtained using the following

equation:

E 

 L    a    b  * 2

* 2

* 2

where ∆L*, ∆a*, ∆b* are the differences between the color values of the protein films and the protein-BC films. Light transmission and opacity of the samples were measured according to the method described by Abdollahi, Alboofetileh, Behrooz, Rezaei and Miraki [27]. The films were cut into a rectangular specimen and placed directly in the spectrophotometer test cell using air as reference. The light transmittance was measured by scanning the samples at wavelengths range 400 to 800 nm using a UV– visible spectrophotometer (UV–2550, Shimadzu, Japan). The samples were measured in triplicate. Opacity of the films was recorded at 600 nm as follows: Opacity = A 600 /X Where, A600 is the absorbance at 600 nm, X is the film thickness (mm). 2.14. Mechanical properties Film samples were conditioned for 2 days in a thermo-hygrostat at 50% RH and 25 °C before measurement. Tensile strength (TS) and elongation at break (EAB) of the films were measured by a texture analyzer (XT PLUS/50, Stable Micro Systems Ltd.) according to ASTM 1997. Five samples (1.8 × 10 cm) of each film were evaluated. Thickness was determined at 10 points using a micrometer with accuracy 0.01mm. The film specimens were stretched with 50 mm of initial grip separation and

50 mm min−1 of cross-head speed. The tensile strength (TS) and the elongation at break (EAB) were obtained as follows: Tensile strength (TS) =

N ab

Elongation at break (EAB %) =

L  L0 100 L0

Where N is the maximum force required to break, a is the width (mm) of the sample, b is the thickness of the sample (mm), L0 is the initial length of the sample (mm) and L is the final length of the sample (mm). 2.15. Statistical analysis All results are expressed as mean values ± standard deviation (SD). Analysis of variance (ANOVA, SPSS 20.0, Chicago, IL, U.S.A.) with the Tukey’s post hoc test was applied to assess the effect of BC. The statistical significance was set at P <0.05. 3. Results and discussion 3.1. Microstructure of the biocomposite films The distribution of BC in the DDGS protein matrix was observed at both the surface and in cross-section of composite films using SEM (Fig.1). The surface morphology of the pure protein film (0% BC) was loose and homogenous without any holes [28]. The film with 1.1% BC was homogenous at the surface, but was smoother than the pure protein film. However, the surface of films containing 1.6%, 1.8% and 2.0% BC were denser, with a few pores, and a changed morphology. Similar results were also observed by Su, Huang, Yuan, Wang and Li [29]. A few aggregations were observed in the film with 2.0% BC. The cross-sectional SEM image showed that the internal structure of the pure protein film was also loose. Interestingly, the internal

structure of the 1.1% BC film was denser, which was very different from control film. This may be due to broad dispersal of the fiber filaments in the protein matrix, but the concentration of BC was too low to form a skeletal structure in the film. Therefore, the TS of the 1.1% BC film was significantly lower than the films with 1.6%, 1.8%, or 2.0% BC (Table 3). In cross-section, the composite films gradually became denser with increasing BC concentration from 1.6% to 2.0%, which indicates that BC is compatible with the protein matrix and endows the protein film with rigidity. This was consistent with the mechanical properties of the composite films (Table 3). 3.2. Fourier transforms infrared (FTIR) spectroscopy In order to identify the functional groups and their chemical environments in the polymer films, composite films containing BC (0% to 2.0%) were analyzed by FTIR (Fig. 2). The broad adsorption band in the range of 3200-3500 cm-1 was related to free and bound OH groups in protein and sorbitol [30]. In the 2.0% BC film, the peak shifted to lower wavenumbers (from 3370 to 3367 cm-1), which can be attributed to hydrogen bonding between BC and protein. Nevertheless, the number of hydrogen bonds acting on the hydrogen atom of the C=O group (amide I) decreased, possibly because BC preferentially interacted with the OH group of protein in the 2.0% BC film BC. The typical absorption band in the range of 2850-2980 cm-1 was assigned to C-H stretching. Other absorption peaks were assigned as follows: (i) 1200-1350 cm-1, to a combination of N-H in-plane bending with C-N stretching vibrations (amide III); (ii) 1400-1550 cm-1, to N-H bending (amide II); and (iii) 1600-1700 cm-1, to stretching vibration of C=O and C-N groups (amide I) [31, 32]. There was one main

peak and a small shoulder at 1632 cm-1 and 1655 cm-1, which were assigned to β-sheet and α-helix structures in the protein [33-35]. For the films containing 1.1, 1.6, 1.8, or 2.0% BC, addition of BC made the Amide II peak shift from 1549 cm-1 to 1545 cm-1, which indicated an increase in hydrogen bonds to the protein’s N-H group. These significant changes were consistent with the changes in the mechanical properties that showed that addition of BC improved the TS of the composite films. 3.3. Differential scanning calorimetry (DSC) Thermal stability of the protein and protein-BC films was investigated by DSC. Three endothermic peaks can be seen in the DSC curves (Fig. 3). The first peak, related to glass transition (Tg), was 50.31 °C for the pure protein film and increased with increasing BC content to 50.49, 50.69, 51.98, and 51.75 °C for 1.1, 1.6, 1.8 and 2.0% respectively. A strong interaction though hydrogen bonds between BC and protein matrix restrained the molecular mobility of the proteins, thereby causing an increase in Tg. This could be correlated with the changes seen in the mechanical properties and by FTIR. Oliveira, Makishi, Chambi, Bittante, Lourenço and Sobral [10] reported that reinforcement of castor bean protein films with cellulose fiber increased the Tg from -50.48 °C to -46.33 °C. Similarly, the Tg of gelatin films increased from 66.9 °C to 69.9 °C after addition of BC [16]. Furthermore, a single Tg was observed in both the protein and protein-BC films, indicating compatibility of the protein with the sorbitol and BC [31]. If a component is not compatible, there will be more than one Tg corresponding to various phases [31, 36]. The other two peaks in the DSC curve can be attributed to the melting peak and

degradation peak of albumin. For instance, the DSC curve of whey protein isolate mixed with 50% glycerol had two endothermic peaks, assigned to Tg (46.9 °C) and Tm (168.0 °C) [32]. Su, Huang, Yuan, Wang and Li [29] also pointed out that the decomposition temperature was in the range of 230-250 °C for a blend of soy protein isolate and CMC. In addition, the temperatures of the second and third peaks increased with increasing BC. This may reflect that the thermal stability was improved because of the enhanced intermolecular hydrogen bonds between BC and protein matrix. 3.4. Viscosity analysis The variation in the shear viscosity of the films was measured with increasing shear rate (Fig. 4). The shear viscosity of the pure protein solution was stable with increasing shear rate. This is the typical feature of a Newtonian fluid, whose viscosity is constant and only several times higher than water. Nevertheless, the protein FFS transformed from Newtonian-like fluid into non-Newtonian fluid after addition of BC, through the formation of a three-dimensional network structure between BC and protein matrix. This transformation from Newtonian-like fluid to non-Newtonian fluid is controlled by the BC behavior. Furthermore, viscosity decreased with increasing shear rate. The mixture exhibited an obvious shear thinning behavior, like most suspensions containing polymers of high molecular weight [37, 38]. This result indicated that, as shear rate increased, the intermolecular junctions were disrupted at a rate faster than their rate of reformation, resulting in a decrease in the junction density and hence a drop in the viscosity [39]. This shear thinning behavior showed that the

network structure of the composite film was formed through strong hydrogen bonds between the BC and protein matrix. It has also been reported that addition of cellulose nanocrystals results in a shear thinning behavior over the full range of shear rate [24]. 3.5. Water solubility (WS) and moisture content (MC) A measure of water solubility can generally estimate the water resistance of the films. The WS of the films decreased from 60.08 to 53.91% with increasing BC, indicating that BC addition significantly decreased solubility in water (Table 1). The WS of the DDGS protein-BC films in this study are higher than that in other studies [10, 29]. This could be due to the water-soluble albumin that is the dominant component of the DDGS, resulting in a higher water sensitivity of the composite films. However, both protein and protein-BC films retained their integrity without separation of BC from the protein matrix even after 24 h of water immersion. This indicated that the intermolecular networks were intact, with only a small amount of low molecular weight-peptides and the sorbitol plasticizer dissolving in water [40]. Films with high solubility can be used for packaging of low moisture-products. Of course, the plasticizer plays an important role in WS. In this study, sorbitol might decrease interactions between biopolymer molecules and increase solubility due to its hydrophilic nature. In addition, sorbitol has six hydroxyls, which result in higher water solubility than films using glycerol [41, 42]. It may be of interest in the future to manufacture and test protein-BC films with other plasticizers. The moisture of the films decreased with increasing BC addition. At high BC levels (1.8 and 2.0%), the films had significantly lower moisture content than those with low

levels of BC (0-1.6%) (Table 1). This likely reflects the hiding of the amide groups by the interactions between the BC and protein matrix, which prevented interactions between protein and water through hydrogen bonds [43]. On the other hand, the formation of hydrogen bonds between the protein and BC decreased the number of hydrophilic hydroxyl groups in the BC, thus reducing the ability of the BC to bind moisture [44]. The moisture of these films (1.6-2.0% BC) averaged 11.15 ± 1.36%, which was lower than films composed of castor bean protein and pulp cellulose fibers (14.35 ± 0.57%) in previous studies [10]. 3.6. Water vapor permeability (WVP) Packaging films should limit mass transfer of moisture between the packaged products and the environment. WVP is used to analyze the amount of water permeating through the packaging materials [2], which is a standard method to assess the films as a moisture barrier. The barrier capacity of a packaging film can extend the shelf life of a product, because physicochemical and microbiological deterioration is greatly affected by the moisture levels inside the package [30]. The WVP of the protein-BC films slightly reduced with increasing BC content (Table 1). The protein and protein-BC films had an average WVP value of 6.24 ± 0.75 ×10-10 g m/Pa s m2, which is one order of magnitude lower than protein films prepared from either sorghum distiller’s dried grains (2.46 ± 0.75×10-9 g m/Pa s m2) [22] or brewer’s spent grain protein-chitosan composite films (2.93 ± 0.2×10-9 g m/Pa s m2) [45]. The WVP values of all the films with and without BC were also lower than other protein films, such as whey protein films (6.75 ± 0.23×10-8 g m/Pa s m2) [46] and peanut protein

isolate-gum arabic films (167.9 ± 0.75×10-11 g m/Pa s m2) [47]. Previous studies reported that the addition of BC made the pathways less available for water molecules to pass through the film [48]. The strong hydrogen bonding interactions between the protein and BC will hinder interactions between protein and water and consequently reduce the channels between the molecules within the film and block the shuttling of water molecules. 3.7. Color The color values of the films with and without BC were analyzed (Table 2). The mean lightness (L*), redness (a*) and yellowness (b*) values were 56.05 ± 3.01; 18.64 ± 0.72; and 27.62 ± 1.28, respectively. There was no significant difference between the different samples (P >0.05), indicating that the BC did not change the optical properties of the films. The values of L*, a*, b* appear to be high for films typically used in packaging materials. The buckwheat DDGS were dark in color, which could explain the higher a*, b* and lower L* of the films in our study. These films may find preferential use as dark packaging materials or for use in the agricultural domain. 3.4. Transparency properties and opacity of films The optical transparency and opacity of the protein films containing BC (0%-2.0%) were investigated using spectroscopy in the visible light region (400-800 nm). The transmittance values of the films exhibited a decreasing trend with increasing BC content (Fig. 5A), results that agree with the observation of Fernandes, Oliveira, Freire, Silvestre, Neto, Gandini and Desbriéres [15]. This result could be explained by

the compatibility of the BC particles and the protein matrix. In general, the transparency was low, because the protein-based films appear to be slightly yellowish (Fig. 5B). Measurement of opacity is an acceptable parameter for the analysis of the transparency of a film. That is to say, higher opacity indicates lesser transparency [25]. It is surprising that addition of BC slightly increased the opacity of the protein films at wavelength of 600 nm (Fig. 5C), although this does agree with the films’ color (Table 2). 3.8. Mechanical properties The addition of BC to the protein films increased the tensile strength of the film (Table 3). The TS of the 1.6%, 1.8% and 2.0% BC-films were significantly higher than that of control (0% BC), demonstrating that BC had a strong positive impact on the mechanical properties of the films. The TS increased by 72% for the 2.0% BC film as compared to control. This is also further evidence of the equal dispersion of BC in the protein matrix [49]. Similar behavior has been reported for castor bean protein films with pulp cellulose fiber (12.5%), which showed increasing TS from 8 to 13 MPa, although the TS of DDGS protein film containing 1.6% BC was higher than that containing 12.5% cellulose fiber [10]. Furthermore, the TS of soy protein films reinforced with cellulose fibers (1%, 5%) increased from 4.7 to 10.83 MPa at 43% RH, with TS in inverse proportion to RH [35]. For our DDGS protein-BC films , the increasing TS could be interpreted as even dispersion of BC in the protein matrix, with intermolecular hydrogen bonds between amide groups in the protein and hydroxyl group of the BC transferring the stress on

BC to the protein matrix [50]. The molecular chain structure of the BC enhanced the internal rigid frame structure of the protein films. On the other hand, addition of BC produced a negative influence on the elongation ability of the films. The elongation at break (EAB) decreased significantly from 21.71 to 7.47% with the increase in BC concentration, demonstrating that the hydrogen bonds tended to be firm and restricted the relative motion of the protein chains. Although the EAB value of the films was low, they were comparable in magnitude with those (EAB from 7 to 10%) reported by Yang, Lee, Won and Song [22]. 4. Conclusion In this study, biocomposite films based on soluble protein from buckwheat distiller’s dried grains (DDGS) were manufactured and reinforced with bacterial cellulose (BC). Use of protein from a waste product is an environmentally friendly investment, and the extraction was done without toxic organic compounds. BC can disperse well in protein matrix through intermolecular interactions, mostly hydrogen bonds, resulting in good compatibility and high quality of the films. Consequently, addition of BC can improved the typical characteristics of protein films, such as water solubility, moisture content, tensile strength and thermal stability. Furthermore, composite films in our study had relatively lower water vapor permeability compared to films based on other waste products reported in other studies, including sorghum distiller’s dried grains protein, brewer’s spent grain protein, and other proteins, such as whey protein, peanut protein. These results showed that addition of BC improves the properties of protein-based biocomposite films, with optimized films containing

1.8% to 2.0% BC. With this study, we hope to promote the use of BC and protein from distiller’s dried grains to produce degradable composite films that have potential for use as packaging, sugar-coating, egg-coating, or other consumer or agricultural products. Acknowledgments We would like to thank Dr Anita K. Snyder, Donald Danforth Plant Science Center, America for her helpful advices and assistance with the English language. This work was supported by the National Natural Science Fund for Young Scholars (Grant No.: 31601497; 31401515), the Shaanxi Province Science and Technology Research and Development Project (Grant No.: 2015NY022), the China Postdoctoral Science Foundation Project (Grant No.: 2016M591857), the National Center of Meat Quality and Safety Control (Nanjing Agricultural University; Grant No.: M2015K06; M2016K01), the Chinese Universities Scientific Fund (Grant No.: 2452015062) and the Earmarked Fund for Modern Agro-industry Technology Research System, China (Grant No.: nycytx-42-G5-01). All authors read, commented on, and approved the final manuscript. Reference [1] J.W. Rhim, H.M. Park, C.S. Ha, Bio-nanocomposites for food packaging applications, Prog. Polym. Sci. 38(10-11) (2013) 1629-1652. [2] T. Janjarasskul, J.M. Krochta, Edible packaging materials, Food Sci. Technol. 1 (2010) 415-448. [3] N. Reddy, Y. Yang, Thermoplastic films from plant proteins, J. Appl. Polym. Sci. 130(2) (2013) 729-738.

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Captions: Fig.1. SEM surface (A1-E1) and cross section morphologies (A2-E2) of protein with 0%, 1.1%, 1.6%, 1.8%, 2% BC Fig.2. FTIR spectra of buckwheat distiller’s dried grains protein films with different percent of BC Fig.3. DSC curves of buckwheat distiller’s dried grains protein films with different percent of BC Fig.4. The effect of BC on viscosity of buckwheat distiller’s dried grains protein film-forming solution Fig.5. UV-vis transmittance (A), digital images (B) and, opacity of 0%, 1.1%, 1.6%, 1.8%, 2.0% BC films (C)

Fig.1.

Fig.2.

Fig.3.

Fig.4.

A

B

C

Fig.5.

Table 1 Moisture content, water solubility and WVP of buckwheat distiller’s dried grains protein film reinforced with different concentration of BC Thickness BC (%)

WVP MC (%)

WS (%)

(mm)

(10-10g m/Pa s m2)

0

0.081±0.004a

15.08±2.72a

60.08±1.62a

6.68±0.78a

1.1

0.079±0.004a

14.54±2.23a

54.66±1.20b

6.34±0.77a

1.6

0.080±0.003a

12.42±0.64a

56.99±1.07b

6.01±0.64a

1.8

0.080±0.002a

7.59±0.41b

56.96±0.96b

6.22±1.01a

2.0

0.076±0.005a

6.11±0.82b

53.91±2.71b

5.95±0.54a

Table 2 Color of buckwheat distiller’s dried grains protein film with different concentration of BC BC (%)

L*

a*

b*

∆E

0

56.90±3.16a

18.47±0.38a

27.15±0.30a

-

1.1

54.57±2.82a

18.50±0.34a

26.70±0.99a

3.26±1.25a

1.6

56.97±2.71a

18.36±0.58a

27.15±1.01a

2.29±0.92a

1.8

56.37±4.65a

18.81±1.42a

28.11±2.45a

4.56±0.63a

2.0

55.43±1.71a

19.04±0.89a

28.98±1.64a

2.72±2.01a

Table 3 Tensile strength (TS) and Elongation at break (EAB) of buckwheat distiller’s dried grains protein film with different concentration of BC BC (%)

Tensile strength(MPa)

Elongation at break (%)

0

4.26±0.66a

21.71±3.74a

1.1

7.71±1.52a

12.90±2.47b

1.6

13.3±1.56b

12.35±0.40b

1.8

14.98±0.97b

7.18±1.82b

2.0

15.03±2.04b

7.47±1.18b

1