corn starch films

corn starch films

Accepted Manuscript Title: Preparation and properties of zein–rutin composite nanoparticle/corn starch films Authors: Shuangling Zhang, Haiyan Zhao PI...

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Accepted Manuscript Title: Preparation and properties of zein–rutin composite nanoparticle/corn starch films Authors: Shuangling Zhang, Haiyan Zhao PII: DOI: Reference:

S0144-8617(17)30433-2 http://dx.doi.org/doi:10.1016/j.carbpol.2017.04.044 CARP 12232

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

21-1-2017 28-3-2017 18-4-2017

Please cite this article as: Zhang, Shuangling., & Zhao, Haiyan., Preparation and properties of zein–rutin composite nanoparticle/corn starch films.Carbohydrate Polymers http://dx.doi.org/10.1016/j.carbpol.2017.04.044 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.

Preparation and properties of zein–rutin composite nanoparticle/corn starch films

Shuangling Zhang*, Haiyan Zhao

Food Science and Engineering College, Qingdao Agricultural University, No. 700 Changcheng Road, Qingdao 266109, P. R. China

*

Corresponding author: Tel.: 086-0532-86080771; fax: 086-0532-86080771

E-mail addresses: *

Shuangling Zhang: [email protected]

Haiyan Zhao: [email protected]

Highlights 

Zein–rutin composite nanoparticle (RN) and corn starch (CS) films were prepared.



Tensile strength and elongation at break increased with increasing RN loading.



RN incorporation decreased the film water vapor permeability and water solubility.



Prepared RN–CS films showed sustained release and strong antioxidant activity.



Cumulative amount of released rutin was 27.1–36.9% of total rutin after 12 h.

Abstract: Edible active films were prepared based on zein–rutin composite nanoparticles (RNs) and corn starch (CS). RNs (0, 0.5, 1, 2, 4, and 10%, w/w) were incorporated into CS films, to act as a natural antioxidant. Scanning electron 1

microscopy and Fourier-transform infrared spectroscopy were used to examine the morphology and functional group interactions. The tensile strength and elongation at break increased from 1.19 to 2.42 MPa, and from 42.10 to 78.84%, respectively, with increasing RN loading. The incorporation of RNs led to the formation of a net-like structure, which decreased the water vapor permeability and water solubility of the RN–CS film. The cumulative amount of rutin detected in vitro after 12 h was 27.1–36.9% of the total amount of rutin. The RNs dispersed in the CS film provided controlled release of rutin. The antioxidant capacities of the films were greater than that of the pure CS film.

Keywords: Edible active film; zein–rutin composite nanoparticle; antioxidant activity; controlled release

1. Introduction The lipid oxidation of unsaturated fatty acids is the main reason for the short shelf-lives and reduced quality of high-fat or fried foods. Lipid oxidation in foods can be reduced by using packaging that inhibits oxygen permeation. This can be achieved using high-oxygen-barrier packaging films or food-contact edible films. Many functional food ingredients and nutraceuticals also contain highly oxidative compounds. Such oxidation can be retarded by adding antioxidants, which delay oxidation onset and/or slow the oxidation reaction. The direct use of antioxidants has limitations because they can be neutralized, evaporated, or rapidly diffused into the food bulk, reducing their effective surface concentration (Pranoto, Rakshi, & Salokhe, 2005; Ouattara, Simard, Piette, Bégin, & Holley, 2000). A current trend with good potential involves incorporating additives into food packaging films or coatings, thereby improving their functionality (Cagri, Ustunol, & Ryser, 2001; Li, Liu, Zhao, & Bai, 2006). The active agents in the film or coating matrix are slowly released to the food surface, where they remain at high concentrations for an extended period (Chollet, Sebti, Martial-Gros, & Degraeve, 2008). Combining antioxidants with packaging films or coatings to reduce the oxygen 2

concentration in the packaged food (and consequently retard lipid oxidation) significantly increases the shelf-life and improves food quality (Quintavalla, & Vicini, 2002). The use of antioxidant-loaded edible films for preventing oxidation of various foods has attracted attention. The incorporation of derivatives from natural sources such as oregano, rosemary, and garlic oils into edible films can improve the antioxidant characteristics of films (Ouattara, Simard, Piette, Begin, & Holley, 2000; Pranoto, Rakshi, & Salokhe, 2005; Seydim, & Sarikus, 2006; Liu, Li, Chuan, Yuan, & Chen, 2016). Adding antioxidants directly to packaging may affect the physical properties of packaging materials. Many also have limited aqueous solubility and degradation. The properties of natural extracts incorporated into materials affect the material functions. The development of nano-biocomposites is an important advance in the materials industry. Nano-biocomposites consist of nano-sized reinforcing fillers dispersed in polysaccharide, protein, or lipid biopolymer matrices (Xie, Pollet, Peter, & Luc, 2013). Zein is the most abundant protein in corn. Various methods can be used to obtain nanoparticles or bioactive-ingredient-loaded nanoparticles from zein. Zein is a good source of natural biopolymeric colloidal particles. Zein has been used to produce spherical nanostructures to encapsulate, protect, and control the release of active ingredients (Ravishankar, Zhu, & Jaroni, 2010). Zein nanoparticles have been loaded with curcumin (Patel, Hu. Tiwari, & Krassimir, 2010), vitamin D3 (Luo, Teng, & Wang, 2012), lutein (Hu, Lina, Liu, Lia, & Zhao, 2012; Kamil Alison, et al.,2016), and quercetin (Patel, Heussen, Hazekamp, Drost, & Velikov, 2012). Zein nanoparticles reinforce biodegradable polymers by interacting with the polymer matrix, improving their mechanical and barrier properties (Cao, Chen, Chang, Muir, & Falk, 2008; Cao, et al., 2008; Kaushik, Singh, & Verma, 2010; Lu, Weng, & Cao, 2006; Mathew, Thielemans, & Dufresne, 2008; Svagan, Hedenqvist, & Berglund, 2009). Polymers loaded with zein nanoparticles therefore have a broad range of potential applications, including use in antioxidant- and antimicrobial-loaded edible films. We prepared edible films from corn starch (CS) and glycerol, and incorporated 3

zein–rutin composite nanoparticles (RNs). The resulting films had a cohesive matrix. Rutin was incorporated into the films via the RNs, giving antioxidant-loaded edible films. The mechanical, physical, and antioxidant properties of the RN–CS films were investigated.

2 Materials and methods 2.1 Materials Native CS with an amylose content of 26.33% (molecular weight: 5 × 104 to 6 × 104) was supplied by the Fulu Food Corp., Ltd. (Tianjin, P. R. China). Glycerol (analytical grade) was used as a plasticizer. Rutin, zein (molecular weight: 2.5 × 104 to

4

×

104),

2,2-diphenyl-1-picrylhydrazyl

(DPPH),

and

2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonic acid (ABTS) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Ethanol, sulfuric acid, potassium phosphate, ammonium molybdate, and potassium persulfate (all analytical grade) were obtained from the Basifu Chemical Corp., Ltd. (Tianjin, P. R. China). Water purified using a Milli-Q system (ZMQ-S5V001, Millipore, Billerica, MA, USA) was used in all experiments.

2.2 RN preparation RNs were prepared using the method reported by Alves et al. (Alves, dos Reisb, Menezesa, Pereirac, & Pereiraa,2015) with modifications reported by Zhang et al. (Zhang, et al., 2014). Rutin (0.1 g) and powdered zein (1.0 g) were dissolved in 80% (volume fraction) aqueous ethanol (20 mL). This stock solution was diluted with water (50 mL) containing 20 mg mL−1 sodium caseinate under continuous stirring (1000 rpm). The solvent was removed under reduced pressure for 10 min at 45 °C. The residue was centrifuged at 4000 rpm for 10 min. The resulting dispersion was freeze-dried for 24 h (FD-1000, EYELA, Tokyo, Japan), yielding RNs.

4

2.3 Methods 2.3.1 RN–CS films A schematic diagram of the preparation of the antioxidant-loaded edible films is shown in Fig. 1. CS (5.0 g) and glycerol (3.0 g) were added to water (80 mL) to obtain a composite solution. RNs were dissolved in water (20 mL) with stirring until a clear solution was obtained. The RN and CS/glycerol solutions were mixed. The RN loading was 0, 0.5, 1, 2, 4, or 10 wt%, relative to dry CS. The mixtures were magnetically agitated for 20 min, and then heated at 100 °C in a water bath for 20 min to completely gelatinize the CS. The solutions were then cooled to room temperature and degassed under a vacuum of 0.15 MPa for 10 min. Approximately 20 g of each mixture were then spread evenly across Petri dishes (diameter 9 cm), which were heated at 45 °C for 8 h in a ventilated oven to remove residual solvent. Films of homogeneous appearance and thickness approximately 0.1–0.2 mm were obtained. The dried starch films were stored in a humidity chamber (20 °C, relative humidity 50%).

2.3.2. Film characterization (1) Morphology The RN–CS films were examined using scanning electron microscopy (SEM; JSM840 microscope, Topcon Corp., Japan). Samples of thickness <2 mm were fixed on stubs, using double-sided adhesive tape, and sputter-coated with gold before observation, to prevent charging. (2) Fourier-transform infrared (FTIR) spectra of RN–CS films FTIR spectra were recorded using a Thermo Nicolet Nexus IS10 spectrophotometer (Nicolet, Wisconsin, USA). Samples were prepared by mixing 1 mg of finely powdered RN–CS film with 100 mg of KBr (Sigma-Aldrich). The mixture was pressed to yield a transparent pellet. FTIR spectra (100 accumulated scans) were recorded at a 4 cm‒1 resolution in the range 4000‒400 cm‒1. (3) Film thickness The film thickness was measured using a digital micrometer. Ten measurements 5

were performed at random film locations; the reported values are the averages of these measurements. Results are expressed in micrometers. (4) Mechanical properties The mechanical properties of the films were investigated using a texture analyzer (TA.XT Plus, Stable Micro System Ltd., Surrey, UK). Tensile strength (TS) and elongation at break tests were performed using RN–CS films cut into 100 × 10 mm2 strips. The TS was calculated by dividing the maximum strength by the film cross-sectional area (film width × initial thickness). The elongation at break (%) was calculated by dividing the extension differential by the initial distance between grips. Before the tests, the samples were water balanced in a humidity drier (20 °C, 50% relative humidity) for 48 h. Ten samples were tested; the reported values are the averages of these measurements. (5) Optical properties The film transparencies were determined based on their absorption at 600 nm, using an ultraviolet-visible spectrophotometer (TU-1810DASPC, Beijing Purkinje General Co., Ltd., Beijing, P. R. China), according to the method described by Maran et al. (Maran, Sivakumar, Sridhar & Thirugnanasambandham, 2013). Film samples were cut into 10 × 40 mm2 strips, and placed directly in the spectrophotometer cuvette. Air was used as the reference. The opacity was expressed as absorbance per unit thickness. Measurements were made in triplicate; the average values are reported. (6) Water solubility The water solubilities of the films were determined using the method reported by Romero-Bestida (Romero-Bestida, et al., 2005). The films were cut into 10 × 40 mm2 strips and stored in a desiccator with silica gel (0% relative humidity) for 7 days. The samples were then accurately weighed, and placed in beakers containing 40 mL of water. The samples were constantly agitated for 24 h at room temperature (approximately 25 °C). The pieces of film that remained after soaking were removed using filter paper (Whatman no. 1) and dried at 60 °C in an oven overnight to achieve a constant weight. Measurements were performed in triplicate. The percentage of total soluble matter (% solubility) was calculated as (initial dry weight − final dry 6

weight)/initial dry weight × 100. (7) Water vapor permeability (WVP) The WVPs of the RN–CS films were determined gravimetrically (Gontard, Duchez, Cuq, & Guilbert, 1994) at a constant relative humidity of 75% at 25 °C, using a modification of the ASTM standard method E 96-80 (1990). The test films were sealed in circular cups of diameter 3 cm and height 4 cm, which contained anhydrous calcium chloride. The test films were fixed to the openings of the cups using paraffin. The cups were placed in desiccators at a relative humidity of 75%. The desiccators were equipped with fans to eliminate stagnant air above the cups. The cell weight was recorded periodically to determine the stationary-state water-vapor transfer. The WVP (g mm−1 s−1 KPa−1) was calculated as: WVP 

m  d , A  t  p

(1)

Where △m is the weight increase of the permeation cell (g), A is the exposed film area (7.1 × 10‒4 m2), △t is the change in time (s), d is the film thickness (mm), and △p is the vapor partial pressure (KPa) difference across the film. The WVP values are reported as averages of eight measurements taken from two different samples (Barbara, Donatella, & Alessandro, 2006). (8) Cumulative release in vitro of loaded rutin The cumulative release in vitro of loaded rutin represents the controlled release of rutin from the RN–CS film. The RN–CS film (0.50 g) was placed in water (25 mL), and the mixture was transferred to a dialysis bag with a molecular weight cut-off of 8–14 kDa. The dialysis bag was incubated in water (225 mL) at 25 °C under continuous stirring. Aliquots (3 mL) of supernatant were removed after 0, 1, 2, 3, 5, 7, 9, and 12 h of dialysis, and the absorbance of the supernatant samples at 510 nm were recorded. The cumulative release was determined by integration of these measurements. The concentrations were determined from a rutin calibration curve for the concentration range 0–3.2 µg mL−1. The results show the proportion of total rutin released. The cumulative release of rutin was calculated using the following equation:

7

i 1

Rti (%) 

a n 1

n



250 ai 3

U0

 100%

(2)

where Rti (%) is the cumulative percentage of released rutin at time ti, the time at which the ith sample was removed; ai is the amount of rutin (µg) at sampling time ti in a 3 mL sample; and U0 is the theoretical 100% release from 0.50 g of RN–CS film (Xiao, Davidson, & Zhong, 2011; Xiao, Gmmel, Davidson, & Zhong, 2011). (9) Antioxidant activity The antioxidant activities of the RN–CS films were determined based on three complementary radical-scavenging assays (ABTS, DPPH, and phosphomolybdenum). RN–CS films (0.02 g for the DPPH and ABTS assays, 0.008 g for the phosphomolybdenum assay) were cut and placed in water (10 mL) and the mixture was stirred for 10 min at room temperature. The DPPH assay was based on the method reported by Brand-Williams et al. (Brand-Williams, Cuvelier, & Berset, 1995), which involves neutralization of DPPH free radicals (DPPH•) by antioxidants. A calibration curve was constructed for each RN–CS film solution extract by mixing different volumes of the extract solutions with 0.6 mL of 1.0 mmol L‒1 DPPH ethanolic solution, and then increasing the volume to 10 mL with ethanol. The reaction between the extract solution and DPPH radicals was monitored by measuring the absorbance change at 516.5 nm after reaction for 2 h in the dark at 25 °C. The percentage reduction in the DPPH absorbance was calculated as (A0 − Ai)/A0 × 100, where A0 is the absorbance of the DPPH solution in the absence of the RN–CS film, and Ai is the absorbance of the DPPH solution after reaction for 2 h in the presence of the RN–CS film. The ABTS method is based on that reported by Re et al. (Re, et al., 1999). An aqueous mixture of 7.0 mmol L‒1 ABTS (5.0 mL) and 2.45 mmol L‒1 potassium persulfate (5.0 mL) was used to generate ABTS•+ radical cations in the dark at room temperature. The formation of ABTS•+ took 16 h, and the ABTS•+ radical solution had to be prepared every 2 days because of its high instability. Once prepared, the ABTS •+ solution was diluted to obtain an absorbance at 730 nm of 0.70 ± 0.02. Ethanol (0.6 8

mL, used as a control) or RN–CS film extract solution was mixed with the diluted ABTS•+ solution to give a final volume of 5.0 mL, and the mixture was left in the dark at room temperature. After 6 min, the absorbance at 730 nm was measured. The percentage reduction in the ABTS•+ absorbance was calculated as (A0 − A1)/A0 × 100, where A0 is the absorbance of the ABTS•+ solution without the sample, and A1 is the absorbance of the ABTS•+ solution with the sample. The phosphomolybdenum method is based on that reported by Prieto (Prieto, Pineda, & Aguilar, 1999). A reagent solution was prepared by mixing 0.6 mol L−1 sulfuric acid, 28 mmol L−1 sodium phosphate, and 4 mmol L−1 ammonium molybdate. Tubes containing 0.1 mL of sample solution mixed with 0.9 mL of reagent solution were sealed and incubated in a water bath at 100 °C for 90 min. The tubes were then cooled to room temperature, and the absorbance of each solution at 695 nm was measured against a blank (0.9 mL of reagent solution mixed with 0.1 mL of sample solvent, incubated in the same manner). (10) Statistical analysis Each experiment was repeated at least three times. Mean values and standard deviations (±SD) were calculated for each sample. All data were analyzed and compared using analysis of variance and Duncan multiple range tests (a = 0.05) to determine significant differences; SPSS 13.0 statistical analysis software (Illinois, USA) was used.

3. Results and discussion 3.1 Microscopic examination of films All the films produced had a homogeneous surface with no bubbles or cracks, and good handling characteristics. The film thickness was 190‒210 µm for all RN–CS films. SEM images of the surfaces and cross-sectional micrographs of CS films with and without RNs are shown in Fig. 2. The control film (Fig. 2A) without RNs had a homogeneous structure, which is consistent with a compact arrangement of polymer chains. Adding RNs reduced the film homogeneity; the various surface morphologies are shown in Fig. 2B, C, and D. The surfaces of the films containing RNs were 9

coarser than that of the control film. This suggests that the polymer chain arrangement was disrupted by other structural elements in the matrix. These elements could be free RNs, derived from nanoparticle micelles in the aqueous environment, or nanoparticles of amylose inclusion complexes. Such elements would produce discontinuities of different magnitudes in the amorphous matrix. Slight crystallinity appeared with increasing RN content in the CS films (Fig. 2E, e and F, f). This is induced by the formation of RN aggregates when a high RN content is incorporated into the film. Similar results have been reported previously (Liliana, et al., 2013).

3.2 FTIR spectroscopy FTIR spectroscopy can be used to identify molecules and their functional groups in polymer matrices (Liu & Tang, 2013). The FTIR spectra of RN–CS films with different RN contents (w/w) are shown in Fig. 3I and II. The peaks at 1665 and 3423 cm‒1 are attributed to C=O and O‒H stretching vibrations (Natarajan, Krithica, Madhan, & Sehgal, 2011). The characteristic peak of rutin is the C=O stretching vibration at 1665 cm‒1. This is a prominent peak in the spectra of the RN–CS films in Fig. 3I, B‒G, indicating RN incorporation into the CS films. The characteristic O‒H stretching vibration of rutin is observed as a small shoulder at 3423 cm‒1 in Fig. 3II, G. In the FTIR spectra of the RN–CS films, the O‒H stretching vibration is observed at approximately 3300 cm‒1 (Fig. 3II, B‒F). FTIR spectra can yield information on hydrogen bonding among film components. The O‒H···O stretching peak of the CS film shifted from 3423 to 3300 cm‒1 on incorporation of RNs. This suggests increased hydrogen bonding, because of interactions between the RNs and CS, compared with that in the pure CS film. The O‒H···O stretching peak shifted from 3283 to 3279 cm‒1 with increasing RN content in the films, as shown in Fig. 3II, B and F. This indicates stronger hydrogen bonding among the film components (Sugantha Kumari, Khaleel Basha, & Sudha, 2012). These interactions are responsible for the good compatibility and consistency of the RN–CS films.

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3.3 Mechanical properties of RN–CS films The TS represents the maximum force per cross-sectional area (prior to stretching) that the film can sustain before breaking. The elongation reflects the extendibility of the film. Table 1 shows that the TS and elongation (%) at break of the films increased significantly with increasing RN loading, except for the 0.5% loading. The TS and elongation at break increased from 1.19 to 2.42 MPa, and from 42.1% to 78.8%, respectively. The RNs in the film are thought to increase the TS and elongation by acting as a filler and plasticizer, thereby increasing the flexibility of the polymer chains. These results are consistent with those of Wu et al. (Wu, Wang, & Ge, 2009), who reported that using SiO2 nanoparticles as a filler improved the tensile properties of starch films. The improved mechanical properties can be attributed to the well-dispersed RNs, and the interactions between RNs and CS chains. The latter reduce chain mobility, increasing the rigidity of the RN–CS film. Chivrac et al. (Chivrac, Pollet, Schmutz,& Averous, 2008) reported that filler surface–polymer chain

interactions

significantly

improved

the

mechanical

properties

of

nanocomposites, especially the TS. The interactions also reduced the chain mobility and increased the macroscopic rigidity (Chivrac, Pollet, Schmutz, & Averous, 2008). Nanofillers are efficient matrix reinforcers, but only when well dispersed so that their interfaces with the matrix are maximized.

3.4 Transmittances of RN–CS films The film transmittances are shown in Table 1. Adding RNs significantly decreased (p < 0.05) the CS film transmittances. This suggests that the RNs made the films opaquer and darker, possibly because of the yellowish color of zein. These results are consistent with those of Pelissari et al. (Pelissari, et al., 2012) for films based on cassava starch and chitosan. Similar results based on color assessment were reported by Bourtoom et al. (Bourtoom, & Chinnan, 2008) and Chillo et al. (Chillo, et al., 2008).

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3.5 Water solubilities of RN–CS films The solubilities of edible films indicate their integrity in aqueous environments. A higher solubility indicates lower water resistance. Film solubility is an important factor in the biodegradability of films used in packaging. A low film solubility is beneficial for storage, but a high film solubility is beneficial for cooking food products coated with edible films (Laohakunjit, & Noomhorm, 2004). Table 1 shows that the percentage water solubility was significantly lower (p < 0.05) for all the RN–CS films compared with that of the CS control. Incorporating RNs into the CS film contributed to the formation of a net-like structure of starch in the film, which decreased the water solubility. The lower water solubility could have been caused by hold back of amylose from starch in the film. A less extensive net-like structure in the film would make it susceptible to swelling in water and therefore to disintegration. The results show that the water solubilities of the films did not significantly decrease (p < 0.05) with increasing RN concentration. The water solubility of the CS film containing 2% RNs was the lowest, at 28.9 %, whereas that of the CS film containing 10% RNs was 32.8 %. This is because of inhomogeneous dispersion and aggregation of excess RNs in the films (Wu, Wang & Ge, 2009).

3.6 Effects of RNs on WVPs of films The effect of zein–rutin composite nanoparticles (RNs) on the water vapor permeabilities (WVPs) of the films is shown in Table 1. The WVPs of the RN–corn starch (CS) films generally decreased with increasing RN content. The WVPs of the RN–CS films were 1.54, 1.50, 1.47, 1.43, 1.35, and 1.47 g mm m‒2 h‒1 kPa‒1 for CS films with RN contents of 0, 0.5, 1, 2, 4 and 10, respectively. These results are similar to those of a previous study (Li, Yin, Yang, Tang, & Wei, 2012), which showed that an antimicrobial film derived from thymol-loaded zein sodium caseinate nanoparticles had a WVP of approximately 2.4 g mm m‒2 h‒1 kPa‒1. Incorporating RNs did not significantly increase the water vapor barrier of the film. Only a slight change, from 1.54 for the film containing 0% RNs to 1.35 g mm m‒2 h‒1 kPa‒1 for that containing 12

4% RNs was observed, as shown in Table 1. The decreased WVP at high RN loadings may be related to changes in the starch network structure. Starch may become denser because of RN incorporation, and well-distributed RNs in the CS film could affect water diffusion pathways. The effects of RN incorporation on the film WVPs are consistent with previously reported results (Lopez, et al., 2014; Sun, Xi, Li & Xiong, 2014). However, a high RN loading of 10% destroyed the uniform structure of the CS film, as shown by SEM, which increased the WVP.

3.7 Cumulative release characteristics of RN–CS films Figure 4 shows the cumulative percentages of rutin detected in water as a function of time for RN–CS films with RN contents of 0.5, 1, 2, 4, and 10% w/w. All test films showed first-order release profiles with biphasic kinetic release trends. A burst effect within 2 h was followed by sustained release for up to 12 h. These results are similar to those reported for peppermint oil encapsulated in freeze-dried zein nanoparticles (Chen, & Zhong, 2015). Faster release was observed at the start of hydration, and the cumulative amount of rutin reached 19.8–27.1% after 2 h. This is attributed to weak binding or adsorption between RNs and CS. In the sustained release phase, rutin was released more slowly from the CS films, with only 27.1–36.9% of the total rutin released after incubation for 12 h. This was because of migration of rutin in nanoparticles into the solution. The rutin delivery system consists of nanoparticles with hydrophobic cores and hydrophilic shells. This system has been extensively studied for encapsulating hydrophobic drugs and nutrients through hydrophobic interactions (Luo, Wang, Zhang, Whent, & Yu, 2011; Li, Liu, Huang, & Xue, 2011). We suggest that RNs dispersed in CS films could provide controlled release in aqueous food packaging materials. Compounds such as 5-fluorouracil gave 80% release from zein nanoparticles in 0.5 h, in pH 6.8 phosphate buffer (Lai, & Guo, 2011). The rutin release time for the current CS films was longer because of their hydrophilicities and low water solubilities. A previous study showed that a lower proportion of 13

encapsulated thymol was released from zein capsules (10%, pH 8.0, 144 h). The particles in that study were larger, which hindered thymol migration to the exterior (Xiao, Davidson, & Zhong, 2014). SEM showed that the cumulative proportion of rutin released increased with increasing RN mass ratio in the current CS films. Increasing the RN content led to discontinuities of differing magnitudes in the amorphous CS matrix. RNs easily migrated out of the CS films, therefore more rutin was detected in the aqueous solvent.

3.8 Antioxidant characteristics of RN–CS films The antioxidant capacities of the starch films were evaluated using DPPH, ABTS, and phosphomolybdenum assays. Table 1 show that the antioxidant capacities of all five films were better than that of the pure CS film. There were significant differences (p < 0.05) between the samples and the control. The values obtained for the pure CS film using DPPH, ABTS, and phosphomolybdenum were 11.22%, 10.18%, and 0.411, respectively (Table 1). This is because the control CS film also had some antioxidant characteristics. Increasing the RN content in the CS films improved the antioxidant properties; for example, for an RN content of 10%, the DPPH, ABTS, and phosphomolybdenum assays gave values of 47.17%, 64.54%, and 0.763, respectively. These results show that the RNs acted as a strong antioxidant in the CS films. In our previous research, DPPH, ABTS, and phosphomolybdenum assays of pure RNs gave values of 48.8%, 53.5%, and 0.69, respectively; these values are in accordance with the RN content of the 10% RN–CS film. For pure zein nanoparticles, the values from the DPPH, ABTS, and phosphomolybdenum assays were 22.3%, 24.3%, and 0.56, respectively; these values are in accordance with the RN content of the 0.5% RN–CS film.

4. Conclusions Active biodegradable RN–CS films were prepared and characterized. SEM images showed that the films were dense and homogeneous with some irregularities, depending on the RN content. FTIR analysis indicated a higher degree of hydrogen 14

bonding in the RN–CS films than in the pure CS film. The TS, elongation, water solubility, and WVP increased with increasing RN addition up to 2% w/w. This indicates that RNs improve the strength, flexibility, and barrier characteristics of CS films. RNs dispersed in CS films could potentially enable controlled release in aqueous food packaging. The rutin nanoparticles can act as a strong antioxidant in the CS films, and this activity can be provided by rutin nanoparticles on or near the film surface. The active films showed long-lasting antioxidant activity, which is important for the controlled release of rutin. Future studies should focus on optimizing film formulation, developing processing methods for improving film properties for specific applications, and understanding the impact of nanoparticle-induced changes on the quality and shelf-life of food products.

Acknowledgements We thank Professors Sun and Li (Food Science and Engineering College, Qingdao Agricultural University, P. R. China) for assistance with indicator determination. This research was supported by an Innovation Project (Grant No. 2014) for Agricultural Technology in Shandong Province, and by the National Natural Science Foundation of China (Grant No. 31401574).

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Figure captions

Figure 1 Schematic diagram of preparation of antioxidant-loaded edible films.

Figure 2 SEM images of surfaces and cross-section of zein–rutin composite

nanoparticle/corn starch films with various contents (w/w) of zein–rutin composite nanoparticles: A & a, 0; B & b, 0.5%; C & c, 1%; D & d, 2%; E & e, 4%; and F & f, 10%.

Figure 3 FTIR spectra of zein–rutin composite nanoparticle/corn starch films with various contents (w/w) of zein–rutin composite nanoparticles: A, 0; B, 0.5%; C, 1%; D, 2%; E, 4%; F, 10%; and G, rutin.

Figure 4 Cumulative rutin release (%) as function of time in Milli-Q water. Samples studied were zein–rutin composite nanoparticle/corn starch films with zein–rutin composite nanoparticle contents of 0.5, 1, 2, 4, and 10% w/w.

21

Corn Starch

Glycerol

Composite Solution

Rutin nanoparticle

Mixtures Magnetically agitated 20 min, water bath (100°C) 20 min, 0.15 MPa vacuum degassed 10 min 20 g of mixture spread evenly across Petri dishes Heated, 45 °C for 8 h, ventilated oven Films

Figure 1.

22

a b a

b a a

b a

b a

b a 23

c b a

d

e b a

f b a

Figure 2

24



F E 3278.65

D

T(%)

C B

3279.90 3281.97

A 3282.66

G

3283.15 3283.99 3423.89



3500

3000

2500

2000

Wavenumber (cm-1)

Figure 3.

25

40

Rutin detected(%)

35 30 25 20 15

0.50%

1%

2%

4%

10%

10 5 0

0

1

2

3

5

7

9

12

Time (h)

Figure 4.

26

Table 1 Mechanical and physical properties of zein–rutin composite nanoparticle/corn starch films.*

RNs content /%

Tensile strength/MPa

Elongation /%

Transmittance /%

Water solubility /%

WVP/g.mm-1. s-1.KPa-1

Control

1.19±0.09 a

42.10±3.91 a

70.58±1.40 d

39.37±8.06 b

1.54±0.05 a

11.22±2.67 a

10.18±1.96 a

0.411±0.017 a

0.5

1.21±0.05 a

50.77±5.53 ab

64.51±2.83 c

31.12±2.30 a

1.50±0.06 a

19.67±3.69 b

20.25±2.65 b

0.533±0.037 b

1

1.35±0.05 a

56.59±4.70 bc

61.55±1.64 c

30.95±3.59 a

1.47±0.12 a

23.71±2.30 b

28.95±1.41 c

0.570±0.050 b

2

1.77±0.10 b

78.84±2.25 c

53.57±4.10 b

28.88±0.71 a

1.43±0.13 a

31.73±2.28 c

38.96±3.84 d

0.680±0.037 c

4

2.23±0.11 c

62.42±6.86 b

47.80±1.45 a

31.93±1.88 a

1.35±0.04 a

38.06±3.84 d

48.49±3.72 e

0.723±0.024 cd

10

2.42±0.11 d

56.81±7.15 bc

44.71±1.67 a

32.79±1.55 ab

1.47±0.17 a

47.17±1.93 e

64.54±2.35 f

0.763±0.030 d

DPPH(%) ABTS(%)

Phosphomolybdenum

Letters (a-f) indicate significant (P < 0.05) difference within the same column by pair comparison in Duncan method. * Values are expressed as the means and standard deviation of three measurements.

27