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nanoencapsulated tea polyphenols into fatty food simulants
Food Hydrocolloids 62 (2017) 212e221
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Food Hydrocolloids journal homepage: www.elsevier.com/locate/foodhyd
Controlled-release of tea polyphenol from gelatin films incorporated with different ratios of free/nanoencapsulated tea polyphenols into fatty food simulants Fei Liu a, b, Roberto J. Avena-Bustillos b, Bor-Sen Chiou b, Yue Li a, Yun Ma a, Tina G. Williams b, Delilah F. Wood b, Tara H. McHugh b, Fang Zhong a, * a
State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, Wuxi, 214122, People's Republic of China Western Regional Research Center, ARS, U.S. Department of Agriculture, Albany, CA, 94710, United States
b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 26 April 2016 Received in revised form 24 July 2016 Accepted 1 August 2016 Available online 3 August 2016
Chitosan nanoparticles with different encapsulation efficiencies were incorporated to obtain gelatin films with controlled-release properties. Nanoparticles tended to aggregate as encapsulation efficiency increased, due to the increase in surface tension of film-forming solutions. The addition of nanoparticles increased the compactness and isotherm hysteresis of films, whereas decreased the moisture diffusion. Tea polyphenol released faster and greater from films in 50% ethanol (4 C) than in 95% ethanol (25 C) fatty food stimulant due to the swelling by water. However, controlled-release showed in both simulants, where free tea polyphenol had the largest release followed by 51.3%, 83.3% and 96.9% encapsulation efficiency. The diffusion coefficient in 50% ethanol was 2 orders of magnitude higher than that 95% ethanol (1013 and 1011 cm2/s, respectively). Films maintained their structures in 95% ethanol after 240 h but not in 50% ethanol. These results might open up new designs in long-term protection for fatty foods. © 2016 Elsevier Ltd. All rights reserved.
Keywords: Tea polyphenol Gelatin film Chitosan nanoparticle Controlled-release Fatty food simulant Antioxidant Chemical compounds studied in this article: Chitosan hydrochloride (PubChem CID: 71853) Sulfobutylether-b-cyclodextrin sodium (PubChem CID: 66577045)
1. Introduction Synthetic antioxidants are being substituted by natural antioxidants due to their potential toxicity and carcinogenicity. Tea polyphenol (TP), a mixture of catechins from tea, can inhibit the pez de Dicastillo et al., 2011; Noronha, oxidation of food systems (Lo de Carvalho, Lino, & Barreto, 2014). However, the hydrophilicity restricts their application in oils and the direct addition might cause pez de Dicastillo, unfavorable reactions with food components (Lo Ares Pernas, Castro Lopez, Lopez Vilarino, & Gonzalez Rodriguez, 2013; Liu et al., 2015). Active antioxidant film is a novel alternative to overcome these limitations. They can also provide
* Corresponding author. Tel.: þ86 510 85197876; fax: þ86 510 85197876. E-mail address: [email protected] (F. Zhong). http://dx.doi.org/10.1016/j.foodhyd.2016.08.004 0268-005X/© 2016 Elsevier Ltd. All rights reserved.
continuous release to the food surface where oxidation occurs most lu, 2014). intensively (Arcan & Yemeniciog More recently, the demand for prolonging the shelf life of food has encouraged the development of controlled-release films, where antioxidants release in a slow, but controlled manner, to maintain food quality for a longer time (Chen, Lee, Zhu, & Yam, 2012). The inclusion of antioxidants in the matrix of nanoparticles might be a valid method to control the release process due to the increase in mez-Estaca, Lo pez-de-Dicastillo, tortuosity of diffusion paths (Go ndez-Mun ~ oz, Catal Herna a, & Gavara, 2014). The controlledrelease of active films have been obtained by incorporating nanoparticles, such as zeinsodium caseinate nanoparticles (Li, Yin, Yang, Tang, & Wei, 2012), lecithin nanoliposomes (Wu et al., 2015), and poly ε-caprolactone nanocapsules (Noronha et al., 2014). Chitosan nanoparticles are a potential alternative to encapsulate TP and provide controlled-release properties for films. Also,
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chitosan nanoparticles can improve the thermal mechanical properties of films (Hosseini, Rezaei, Zandi, & Farahmandghavi, 2015a, 2016). In our previous study, TP-loaded chitosan nanoparticles, prepared by the ionotropic gelation between chitosan hydrochloride (CSH) and sulfobutyl ether-b-cyclodextrin sodium (SBE-b-CD), have been successfully incorporated into gelatin films (Liu et al., 2015). The protection effects for sunflower oil within the bags made of films varied with the encapsulation efficiency (EE) of TP in CSH-SBE-b-CD nanoparticles (CSNs). However, the causes of the differences in protection effects were still not clearly know. The differences in release properties of TP from gelatin films were not directly investigated and compared. The release of antioxidants from films is a complex process relating to the polymer matrix, pez-de-Dicastillo, nature of antioxidants and properties of foods (Lo mez-Estaca, Catala , Gavara, & Hern ~ oz, 2012). Go andez-Mun The objective of this study was to compare the release properties of TP from gelatin films, with different ratios of free TP to nanoencapsulated TP, into fatty food simulants. We wanted to confirm if the controlled-release of TP could be obtained by varying the EE of TP without the increase of content, where the free TP would release faster while the encapsulated TP release slower to extend the protection effect. CSNs with different EEs were prepared by adjusting the concentration of CSH. The dispersion of CSNs within the gelatin matrix was examined to determine its influence on the final properties and applications of films (Teodoro, Mali, Romero, & de Carvalho, 2015). The films’ antioxidant properties were investigated by monitoring the release of TP into two different fatty food simulants and their DPPH radical scavenging activity. The physical, morphological and structural properties of films were monitored as well.
hydrating gelatin powders in distilled water for 1 h at room temperature and then heating to 65 C with continuous stirring. Glycerol (20% w/w of gelatin) was added as a plasticizer to reduce the brittleness of films. The gelatin solution (25 mL) was then added to 75 mL of freshly obtained nanoparticle suspension or 75 mL of water/TP-containing aqueous solution (0.25 mg/mL). After stirring for 1 h, 35 mL of the film forming solution was cast in a square Petri dish (10 cm 10 cm) and then dried at room temperature (23 C) in darkness for 2 days until it reached a constant weight. Films were conditioned at 25 C and 53% relative humidity (RH) for at least 48 h before testing.
2. Materials and methods
2.4. Moisture sorption isotherm of films
2.1. Materials
Water vapor adsorption-desorption isotherms were obtained at 25 C on a dynamic vapor sorption (DVS) Advantage apparatus (Surface Measurement Systems NA, Allentown, Pa., USA). Prior to analysis, film samples were dehydrated in a desiccator at 25 C until reaching equilibrium. About 5 mg of the film was loaded in a weighing basket and the change in mass was recorded over a humidity range of 0e98%. The diffusion coefficients were calculated from the adsorption-desorption kinetics according to Park and Crank (1968) using the DVS Analysis software (Version 6.3, Surface Measurement Systems Ltd, UK). The Brunauer, Emmett and Teller (BET) surface area was calculated from the linear part of the BET plot according to IUPAC recommendations.
Gelatin (type B from bovine skin, ~200 Bloom value) and glycerol were purchased from China Medicine (Group) Shanghai Chemical Reagent Co. (Shanghai, China). Chitosan hydrochloride (CSH), with a molecular weight of 100 kDa, a degree of deacetylation of 86% and derived from crab shells, was obtained from Golden-Shell Biochemical Co., Ltd. (Hangzhou, China). Sulfobutyl ether-b-cyclodextrin sodium (SBE-b-CD, food grade), with a molecular weight of 2.082 kDa and an average degree of substitution of 6, was purchased from Kunshan Chemical Industries Co., Ltd. (Jiangsu, China). Tea polyphenol (TP), with a polyphenol content 98% and catechins content 90% (determined by manufacturer), was obtained from Qiangsheng Medicine Science Technology Co., Ltd. (Shanghai, China). 2,2-Diphenyl-1-picrylhydrazyl (DPPH) and gallic acid was purchased from Sigma Chemical Co. (St. Louis, MO, USA). All other reagents were analytical grade. 2.2. Preparation of films Gelatin films embedded with various ratios of free to encapsulated TP were fabricated by incorporating CSNs with three different TP EEs (51.3%, 83.3% and 96.9%) according to our previous studies (Liu, Antoniou, et al., 2016; Liu et al., 2015). First, 1 mg/mL TP was added to 24 mL of 2, 4 or 8 mg/mL SBE-b-CD and the solutions were mildly stirred at room temperature for 24 h to promote the formation of inclusion complexes. These solutions were then added drop-wise to 72 mL of 0.5, 1 or 2 mg/mL CSH solutions with vigorous magnetic stirring, respectively. The blank CSNs without TP were also prepared as controls. Freshly obtained CSNs were immediately used to prepare films. Subsequently, 8% (w/v) gelatin solutions were prepared by
2.3. Characterization of film-forming solutions 2.3.1. Scanning electron microscopy (SEM) analysis To observe the dispersion of CSNs within the gelatin matrix, one drop (10 mL) of CSN suspensions and film-forming solutions was placed on the surface of aluminum foil, dried at room temperature and then sputter coated with gold under vacuum. SEM images were obtained using a Hitachi S-4700 scanning electron microscope (Hitachi, Japan) operating at 5 kV. 2.3.2. Surface tension measurement The surface tension of CSN suspensions and film-forming solutions was determined with a Krüss K100 Force Tensiometer (Germany) at room temperature according to Mostafavi, Kadkhodaee, Emadzadeh, and Koocheki (2016) Ultrapure water was used in the calibration before the measurement. The Wilhelmy plate method was used and surface tension was recorded for 120 s after placing the platinum plate on the dispersion surface.
2.5. Release study of films Two food simulants, 95% ethanol and 50% ethanol, were used to perform the release studies. The 95% ethanol can be regarded as a simulant for fats, oil and fatty foods due to their similar hydrophobicity (Noronha et al., 2014). The 50% ethanol can be regarded as a simulant for oil in water emulsions and alcoholic beverages (Ineiguez-Franco et al., 2012). The release of tea polyphenols (TP) from films was determined by adding film pieces (3 3 cm) and 30 mL of food simulants into 50 mL glass vials with screw caps containing a PTFE/silicon septum. Release tests were conducted in the dark at 25 C for 95% and 4 C for 50% ethanol simulants, respectively. The food simulant (0.1 mL) was collected and its TP content was determined at predetermined times (0, 1, 2, 4, 6, 8, 10, 24, 48, 72, 96, 144, 168, 192, 216, 240 h). Simulants were deoxygenated with bubbling nitrogen and the vials were flushed with nitrogen before closing. All analyses were measured in triplicate with three different vials. The release kinetics when the released TP amount was lower than 0.6 of initial TP
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loading were calculated according to:
Mt 4 Dt ¼ Lp p Mp
1=2 (1)
where Mt was the mass of TP in the food simulants at a particular time t (s), MP was the initial TP loading in the gelatin films, Lp was film thickness and D was the diffusion coefficient. D was determined from the slope of the plot of Mt/Mp as a function of t1/2 (Arrieta et al., 2014; Chen et al., 2012).
2.5.1. Total phenol assay The amount of TP released from films was measured by using the Folin-Ciocalteu method (Yu, Tsai, Lin, Lin, & Mi, 2015). Food simulant (20 mL) was thoroughly mixed with 100 mL of FolinCiocalteu reagent (10% v/v). Then, 80 mL of 7.5% (w/v) sodium carbonate was added to the mixture and the sample was allowed to stand for 60 min at room temperature. The absorbance was measured at 725 nm in a Microplate Reader (Shimadzu UV-2450, Japan). Total phenol content was expressed as mg gallic acid equivalent/g film sample according to:
Fig. 1. SEM micrographs of selected CSN suspensions (magnification: 1000 and 10,000) and film-forming solutions (magnification: 10,000) dried on the surface of aluminum foil. xCSH-TP, TP-loaded CSNs prepared at the indicated CSH concentrations. Gel-TP and Gel-xCSH-TP, films prepared with gelatin þ TP-loaded CSNs at the indicated CSH concentrations (mg/mL).
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Total Phenol Content ¼ ðC V Þ=M
(2)
where C was the concentration of gallic acid obtained from the standard curve (mg/mL), V was the volume of the food simulant (mL) and M was the weight of added film (mg). 2.5.2. DPPH radical scavenging activity A previously described method was used to evaluate the DPPH radical scavenging activity (Liu et al., 2015), with slight modifications. Food simulant (80 mL) was mixed with 100 mL (0.1 mM) of DPPH dissolved in ethanol. The mixture was kept in the dark for 30 min at room temperature. The absorbance was measured at 517 nm in a Microplate Reader (Shimadzu UV-2450, Japan). The assay was carried out in triplicate. The DPPH radical scavenging activity was determined according to:
DPPH scavenging ð%Þ ¼ 100 ð1 ðAs Ab Þ=Ac
(3)
where As was the sample absorbance after addition of food simulant, Ab was the absorbance of the food simulant and Ac was the absorbance of the control sample containing blank food simulant mixed with the DPPH solution. 2.6. Film characterization during release study 2.6.1. Swelling tests Swelling degree (SD) of films was monitored during release studies in the food simulants according to the method of Yu et al. (2015). Film samples (3 3 cm) were previously dried and weighted before immersion into 30 mL of the food simulants. At selected times (0, 2, 10, 24, 48, 72, 96, 144, 168, 192, 240 h), film samples were removed from the glass vials and carefully wiped of excess liquid. The weight was measured and the film samples were returned to the vials. SD was calculated from the initial weight of dried film samples (m0) and the weight of swollen films at each time (mt), using:
SD ð%Þ ¼ 100 ð1 þ ðmt m0 Þ=m0 Þ
(4)
2.6.2. Scanning electron microscopy (SEM) and X-ray diffraction (XRD) Film samples were taken out from the glass vials and dried at ambient temperature after 0, 24, 96, 240 h exposure to the food simulants. The cross sections of films were characterized with a scanning electron microscope (SEM, S-4800, Hitachi, Japan) under high vacuum mode at an accelerating voltage at of 2 kV. Film pieces were cryofractured after freezing in liquid nitrogen to obtain the cross sections. Samples were mounted on the specimen holder using double-sided adhesive tapes and then gold coated with a sputter coater (Emitech K550X, Quorum Technologies Inc., UK) under 15 mA for 45 s. The changes in crystalline phases of gelatin films during release tests were determined by XRD. XRD patterns of films were obtained by an X-ray diffractometer (D8 Phaser, Bruker AXS Germany) with Cu Ka radiation (1.542 Å) operating at 40 kV and 40 mA. Data were recorded in the range (2q) of 3e60 with a step size of 0.02 and a scanning rate of 4 min1.
215
test was used to determine the significant differences of the mean values (P < 0.05). 3. Results and discussion 3.1. Dispersion of CSNs within film matrix As shown in Fig. 1, individual nanoparticles could be identified in CSN suspensions at low CSH concentration (0.5 mg/mL). However, particles became aggregated with an increase in CSH concentration and the spherical shape was also changed at 2 mg/mL of CSH. This aggregation might be due to the enhanced hydrogen bonding interactions between CSNs at higher concentrations during the drying process (Hosseini, Rezaei, Zandi, & Farahmandghavi, 2015b). A smooth and homogeneous surface morphology was observed for the control film (Gel-TP). The incorporation of CSNs into the film-forming solution led to the development of a rougher surface. At low CSH concentration (0.5 mg/mL), CSNs appeared to be dispersed evenly within the gelatin matrix. Also, the agglomeration between CSNs in the films occurred with an increase in CSH concentration. CSNs collided and aggregated in the gelatin matrix during drying and the frequency of collision increased at higher CSH concentrations (Hosseini et al., 2016; Wu et al., 2015). However, a denser and more compact surface structure was obtained at higher CSH concentrations due to strong bonding between CSNs and gelatin. Similar results were also reported by Hosseini et al. (2015a) in the fabrication of chitosan nanoparticle composite gelatin films. 3.2. Surface tension of CSN suspensions and film-forming solutions Surface tension can be used to better understand the formulation and processing of edible films or coatings, including interactions between components, wetting or encapsulation (Karbowiak, Debeaufort, & Voilley, 2006). Samples with gelatin or higher CSN concentrations took longer to reach their equilibrium surface tension value. This is shown in the plot of surface tension as a function of time in Fig. S1. The presence of CSNs caused a decrease in surface tension values (P < 0.05) in both CSN suspensions and film-forming solutions (Fig. 2). This could be attributed to the lower surface tension of chitosan solution (61.8 mN/m) compared to that lezof TP solution (71.4 mN/m) (Vargas, Albors, Chiralt, & Gonza
2.7. Statistical analysis Data were presented as mean value ± standard deviation. The data were analyzed by one-way analysis of variance (ANOVA) using the SPSS 19.0 package (IBM, New York). Duncan's multiple range
Fig. 2. Surface tension of CSN solutions and related film-forming solutions. TP and xCSH-TP, TP/CSN suspensions and corresponding film-formation solutions prepared at the indicated CSH concentrations (mg/mL).
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Martínez, 2009). More CSNs absorbed on the surface of CSN suspensions at higher CSH concentrations, leading to the further decrease in surface tension. The 0.5 mg/mL CSH film-forming solution had the lowest
surface tension. The addition of gelatin could change the CSH structure and distribution of CSNs within the solutions due to interactions between CSNs and gelatin (Mostafavi et al., 2016). Furthermore, glycerol tended to reduce the surface tension of filmforming solutions (Caro et al., 2016; Wexler & Dutcher, 2013). An optimum dispersion of CSNs in the film-forming solutions as well as the lowest intermolecular forces between CSNs, gelatin and glycerol might have led to the lowest surface tension for the 0.5 mg/ mL CSH sample (Qun & Ajun, 2006). According to Mostafavi et al. (2016), the suspension spreadability and coating integrity critically depended on the surface tension of solutions. Therefore, higher surface tensions induced by the increased CSH concentrations might have led to the aggregation of CSNs and the formation of beads within the gelatin matrix (Fig. 1). 3.3. Moisture sorption isotherm and diffusion coefficient Equilibrium moisture contents of films varied with the water vapor pressure surrounding them. The adsorption and desorption isotherms of all films had a sigmoidal shape (Fig. 3a, b), showing an asymptotic trend as water activity (aw) approached 1 (Suppakul, Chalernsook, Ratisuthawat, Prapasitthi, & Munchukangwan, 2013). The incorporation of CSNs resulted in films with lower equilibrium moisture contents, especially at aw > 0.6. Similar
Fig. 3. Moisture (a) adsorption and (b) desorption isotherms of gelatin films. (c) Hysteresis values of adsorption and desorption isotherms. Gel-TP and Gel-xCSH-TP, films prepared with gelatin þ TP-loaded CSNs at the indicated CSH concentrations (mg/mL).
Fig. 4. Diffusion coefficients of (a) adsorption and (b) desorption process of gelatin films as a function of target relative humidity. Gel-TP and Gel-xCSH-TP, films prepared with gelatin þ TP-loaded CSNs at the indicated CSH concentrations (mg/mL).
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results were obtained by Teodoro et al. (2015) after incorporating acetylated starch nanoparticles into cassava starch films. The addition of CSNs resulted in CSN-gelatin interactions, leading to a reduced number of polar sites available for hydrogen bonding with water molecules (Erbas, Aykin, Arslan, & Durak, 2016). The hysteresis in films, which was related to the nature and state of components (Lemus M, 2011), also reflected the rearrangements of structure and conformation of gelatin in the presence of CSNs (Fig. 3c). The largest hysteresis corresponded to the lowest relaxation rate in the adsorption/desorption process and was observed for the Gel-1CSH-TP sample (Gontard, Guilbert, & Cuq, 1993), which had the most compact microstructure. As shown in Fig. 4, good agreement was observed between the diffusion coefficients and the sorption isotherms. The addition of CSNs resulted in a decrease in the diffusion coefficient values, with the lowest diffusion coefficient obtained for the Gel-1CSH-TP sample in the relative humidity (RH) range of 30e60%., The moisture diffusion coefficient was also dependent on RH, similar to the inz, and McHugh results of Otoni, Avena-Bustillos, Olsen, Bilbao-Sa (2016). The diffusion of moisture occurred by several mechanisms, including adsorption to the monolayer, vapor transport through a swollen matrix and collapse of the structure (Otoni et al., 2016). Moreover, the barrier property of films depended on the moisture diffusion coefficient and its transport within the matrix (Srinivasa, Ramesh, Kumar, & Tharanathan, 2003). Therefore, the diffusion coefficient values could be used to explain the differences in water barrier properties of films. These values correlated well with the WVP data (Table S1). In addition, the volume of vapor monolayer (Vm) and the corresponding specific surface area of gelatin films could be calculated from the adsorption isotherm (aw < 0.45) using the classical BET equation (Table 1). The changes in these values
Table 1 Vm and Specific Surface Area of Gelatin Films Calculated from the BET Model when aw < 0.45.a Films
Vm (mmol/g)
Gel-TP Gel-0.5CSH-TP Gel-1CSH-TP Gel-2CSH-TP
3.3 2.6 2.1 2.9
± ± ± ±
0.1a 0.1c 0.1d 0.1b
Specific surface area (m2/g) 229.1 181.1 143.9 199.2
± ± ± ±
6.9a 5.4c 4.3d 6.0b
Different lowercase letters in the same column indicate a statistically significant difference (P < 0.05). Gel-TP and Gel-xCSH-TP, films prepared with gelatin þ TPloaded CSNs at the indicated CSH concentrations (mg/mL). Vm, the amount of vapor adsorbed if a monolayer was to form. a Values are expressed as mean ± standard deviation.
217
were consistent with the sorption isotherm and diffusion coefficient results. The differences in the specific surface areas might have affected the release of TP from gelatin films. 3.4. Swelling degree (SD) Food simulant resistance and release of active compounds were related to the swelling of films (Garde, Catala, Gavara, & Hernandez, 2001; Yu et al., 2015). Gelatin is a hydrophilic polymer that can swell in contact with water, which would facilitate the diffusion of active compound through its matrix (Buonocore, Del Nobile, Panizza, Corbo, & Nicolais, 2003). Different swelling degrees were observed when the films were placed in 95% and 50% ethanol fatty food simulants (Fig. 5). The gelatin films immersed in 95% ethanol swelled slowly, whereas the films immersed in 50% ethanol swelled more quickly. In 95% ethanol, the films containing CSNs had slightly lower SD. Also, an increase in CSH concentration resulted in a further decrease in SD (Fig. 5a). These results could be due to an increase in intermolecular attractive forces between CSNs and gelatin at higher CSH concentrations, resulting in reduced free volume and a more compact and less permeable film matrix (Hosseini et al., 2015a). In comparison, the addition of CSNs to films led to higher SD in 50% ethanol (Fig. 5b). This might be attributed to the slight hydrophilic nature of CSNs as reported by Ooi, Ahmad, and Amin (2015). The reduced free volume in films with the highest CSH concentrations (2 mg/mL) also restricted the gelatin chain movements and reduced the swelling capability of films in 50% ethanol. 3.5. Release of TP from films into fatty food simulants The release of TP from gelatin films into 50% ethanol fatty food simulants was measured at 4 C, whereas the release into 95% ethanol was carried out at 25 C to monitor the TP release (Fig. 6). In each simulant, all films had similar release profiles with an initial fast release followed by a sustained slow release. The initial fast release of TP provided the short-term protection and the subsequent slower release guaranteed the intermediate/long-term protection (Chen et al., 2012). The total TP release after 240 h could be controlled by the amount of CSNs added to the films. For example, the Gel-2CSH-TP film in both simulants released approximately half of the TP released from the blank films (Gel-TP). Also, the extent of TP release depended on the amount of free TP within the gelatin matrix, which was related to the encapsulation efficiency (EE). The EE could be controlled by the CSH concentration used for CSN
Fig. 5. Swelling degree (SD) of gelatin films immersed into (a) 95% ethanol (25 C) and (b) 50% ethanol (4 C) fatty food simulants as a function of time. Gel-TP and Gel-xCSH-TP: films prepared with gelatin þ TP-loaded CSNs at the indicated CSH concentrations (mg/mL).
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Fig. 6. Release of TP from gelatin films (left) and related DPPH radical scavenging activity of fatty food simulants (right) of (a) 95% ethanol (25 C) and (b) 50% ethanol (4 C) fatty food simulants as a function of time. Inset is the straight line representation of Mt/Mp vs. t1/2 according to equation (1). Gel-TP and Gel-xCSH-TP: films prepared with gelatin þ TPloaded CSNs at the indicated CSH concentrations (mg/mL).
Table 2 Diffusion Coefficient (D) of TP Released from Gelatin Films into 95% (25 C) and 50% Ethanol (4 C) Fatty Food Simulants.a Films
Gel-TP Gel-0.5CSH-TP Gel-1CSH-TP Gel-2CSH-TP
95% Ethanol
50% Ethanol
D 1013 (cm2/s)
R2
D 1011 (cm2/s)
R2
1.59 1.46 0.58 0.38
0.987 0.988 0.985 0.978
5.54 1.19 2.04 0.39
0.969 0.948 0.9303 0.9646
a Gel-TP and Gel-xCSH-TP, films prepared with gelatin þ TP-loaded CSNs at the indicated CSH concentrations (mg/mL).
preparation, which decreased with increasing CSH concentration, according to our previous studies (Liu, Antoniou, et al., 2016; Liu et al., 2015). As shown in the inset graphs of Fig. 6, the model (1) fitted the experimental data well (R2 > 0.9) and the D values are shown in Table 2. The samples in 50% ethanol had D values that were 2 orders of magnitude higher than those in 95% ethanol. The intake of more water in 50% ethanol (see the SD of films in Fig. 5) led to the plasticization and an increase in D values (Buonocore et al., 2003; pez-de-Dicastillo et al., 2012). The addition of CSNs led to a Lo slower release of TP, with the 2 mg/mL CSH sample having the
Fig. 7. The intensity ratio of crystal and amorphous peak (It/Ia) of gelatin films exposed to (a) 95% ethanol (25 C) and (b) 50% ethanol (4 C) fatty food simulants at the selected release times. Gel and Gel-xCSH, films prepared with gelatin þ control or TP-loaded CSNs at the indicated CSH concentrations (mg/mL).
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Fig. 8. SEM micrographs (magnification 10,000) of cross section of gelatin films exposed to (a) 95% ethanol (25 C) and (b) 50% ethanol (4 C) fatty food simulants at the selected times (h). Gel-TP and Gel-xCSH-TP, films prepared with gelatin þ TP-loaded CSNs at the indicated CSH concentrations (mg/mL).
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slowest release. The D values decreased from 1.59 1013 to 0.38 1013 cm2/s and from 5.54 1011 to 0.39 1011 cm2/s in 95% ethanol and 50% ethanol, respectively. The presence of CSNs increased the tortuosity of the TP diffusion path and modified the compactness of the gelatin film structure, thus hindering the mez-Estaca et al., 2014; Liu et al., 2013). The release release of TP (Go of TP from Gel-1CSH-TP film was faster than that from Gel-0.5CSHTP film in 50% ethanol (Fig. 6b), which is different from that in 95% ethanol fatty food simulants. This difference might be due to the higher SD of Gel-1CSH-TP film in 50% ethanol (Fig. 5b). In addition to the diffusion of TP from film, the release in 50% ethanol also resulted from the diffusion of simulant into film and the relaxation of film matrix (Buonocore et al., 2003). pez-de-Dicastillo et al. (2012), it was necessary According to Lo to verify that the active agents retained their antioxidant activity during the release. DPPH radical scavenging activity was used to determine the antioxidant activity of the samples. As shown in Fig. 6 (right), the DPPH radical scavenging activity was proportional to the released TP concentration in 95% ethanol during all the release times. In comparison, this relationship was only observed before 8 h of release in the 50% ethanol. The differences in DPPH radical scavenging activity between various gelatin films were negligible (P > 0.05) after 8 h of release. This similarity could be due to the presence of radical scavenging gelatin and chitosan once they dissolved in the 50% ethanol (Liu et al., 2015; Wan, Xu, Sun, & Li, 2013; Yen, Yang, & Mau, 2008). 3.6. Changes in gelatin film structure during release study All gelatin films had an XRD pattern characteristic of a partially crystalline material, with a sharp peak at ~7 (2q) and a broad peak at ~20 (2q), which corresponded to the triple-helical crystalline structure and the amorphous fractions, respectively (Fig. S2) (Ge et al., 2015; He, Zhang, Cai, & Wang, 2016; Liu, Majeed, et al., 2016). The XRD patterns of gelatin films with blank CSNs (without TP) were also determined as controls. The presence of TP decreased the intensity of the triple-helix peaks. Also, the CSH and SBE-b-CD peaks in the XRD patterns disappeared after forming CSNs, according to Mahmoud, El-Feky, Kamel, and Awad (2011). Therefore, the decrease in the triple-helix peaks might be due to hydrogen bonding between gelatin and TP (Wu et al., 2013), which inhibited the triple-helix formation. The relative triple-helix content could be expressed as the intensity ratio of crystal to amorphous peaks (It/Ia), as shown in Fig. 7. The incorporation of nanoparticles destroyed the hydrogen bonds between gelatin molecules (He et al., 2016), resulting in a decrease in triple-helix content at higher CSH concentrations. Arrieta et al. (2014) reported that an increase in the amount of crystalline regions resulted in slower diffusion of catechin from poly (lactic acid)-poly (hydroxybutyrate) blend films. However, the opposite trend occurred in our study, with samples of higher crystallinity having faster release rates (Fig. 6, Table 2). Although the CSNs retarded some formation of triple helices, the release of TP was mainly controlled by its diffusion from CSNs. Also, all gelatin films that remained in the simulants for longer times had lower triplehelix contents. This decrease in triple-helix content might be due to the modification and relaxation of gelatin conformation as food simulants diffused into the matrix. Furthermore, some tightly bound water, which was necessary to form and stabilize the gelatin triple-helix (Frazier & Srubar, 2016), might have been displaced by ethanol. The microstructure changes of gelatin films during TP release were examined by SEM. In contrast to the film-forming solutions dried on the aluminum foil surface (Fig. 1), the films had smooth surfaces even at the highest CSH concentration (Fig. S3, 2 mg/mL).
This might be explained by the uniform dispersion of CSNs within the gelatin matrix (Fig. 8). Although samples with higher CSH concentrations showed rougher surfaces, no individual CSNs could be identified in their cross-section micrographs. According to Arrieta et al. (2014), the integrity of films, especially those made of biodegradable polymers, should be maintained during the shelf life of the food. As shown in Fig. 8, all gelatin films became rougher at longer release times, which might be due to the plasticization effect pez-de-Dicastillo et al., 2012). Films in (Buonocore et al., 2003; Lo 50% ethanol had rougher surfaces than those in 95% ethanol. Also, the greater penetration of the 50% ethanol solution into gelatin films (Fig. 5) led to the presence of voids or wrinkles in the surface micrographs (Fig. S3b). Therefore, gelatin films containing CSNs were more suitable for use in oils rather than in oil in water emulsions since their microstructures could be better maintained. 4. Conclusions In conclusion, desirable controlled-release properties of gelatin films were obtained by incorporating different ratios of free to nanoencapsulated TP. The release of TP from films was fatty food simulant- and EE-dependent. The release process was related to the morphological changes. The lower the ethanol ratio, the higher in TP release. The higher release process was accompanied with the loss in film integrity. The higher the EE, the lower the ratio of free to encapsulated TP, and thus, the lower the ultimate release percentage. The presence of CSNs increased the tortuosity of TP diffusion paths into simulants, which further increased by increasing CSH concentration in CSNs preparation. Therefore, the free TP released at a faster rate could maintain the short-term antioxidant properties for fatty foods, whereas the encapsulated TP released at a slower rate could maintain the intermediate and/or long-term antioxidant properties. Acknowledgements This work was financially supported by the National 125 Program 2011BAD23B02, 2013AA102207, National Natural Science Foundation of China 31171686, 31401532 and 31571891, Natural Science Foundation of Jiangsu Province BK2012556, 111 Project B07029, PCSIRT0627, JUSRP11422 and JUSRP51507 and the Selfdetermined Research Program of Jiangnan University, JUSRP 115A22. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.foodhyd.2016.08.004. References lu, A. (2014). Controlled release properties of zeinfatty acid Arcan, I., & Yemeniciog blend films for multiple bioactive compounds. Journal of Agricultural and Food Chemistry, 62, 8238e8246. Arrieta, M. P., Castro-Lopez Mdel, M., Rayon, E., Barral-Losada, L. F., LopezVilarino, J. M., Lopez, J., et al. (2014). Plasticized poly(lactic acid)poly(hydroxybutyrate) (PLA-PHB) blends incorporated with catechin intended for active food-packaging applications. Journal of Agricultural and Food Chemistry, 62, 10170e10180. Buonocore, G. G., Del Nobile, M. A., Panizza, A., Corbo, M. R., & Nicolais, L. (2003). A general approach to describe the antimicrobial agent release from highly swellable films intended for food packaging applications. Journal of Controlled Release, 90, 97e107. pez, L., Abugoch, L., & Tapia, C. (2016). Novel Caro, N., Medina, E., Díaz-Dosque, M., Lo active packaging based on films of chitosan and chitosan/quinoa protein printed with chitosan-tripolyphosphate-thymol nanoparticles via thermal ink-jet printing. Food Hydrocolloids, 52, 520e532. Chen, X., Lee, D. S., Zhu, X., & Yam, K. L. (2012). Release kinetics of tocopherol and quercetin from binary antioxidant controlled-release packaging films. Journal of
F. Liu et al. / Food Hydrocolloids 62 (2017) 212e221 Agricultural and Food Chemistry, 60, 3492e3497. Erbas, M., Aykin, E., Arslan, S., & Durak, A. N. (2016). Adsorption behaviour of bulgur. Food Chemistry, 195, 87e90. Frazier, S. D., & Srubar, W. V. (2016). Evaporation-based method for preparing gelatin foams with aligned tubular pore structures. Materials Science and Engineering: C, 62, 467e473. Garde, J. A., Catala, R., Gavara, R., & Hernandez, R. J. (2001). Characterizing the migration of antioxidants from polypropylene into fatty food simulants. Food Additives and Contaminants, 18, 750e762. Ge, L., Li, X., Zhang, R., Yang, T., Ye, X., Li, D., et al. (2015). Development and characterization of dialdehyde xanthan gum crosslinked gelatin based edible films incorporated with amino-functionalized montmorillonite. Food Hydrocolloids, 51, 129e135. mez-Estaca, J., Lo pez-de-Dicastillo, C., Hern ~ oz, P., Catala , R., & Go andez-Mun Gavara, R. (2014). Advances in antioxidant active food packaging. Trends in Food Science & Technology, 35, 42e51. Gontard, N., Guilbert, S., & Cuq, J. L. (1993). Water and glycerol as plasticizers affect mechanical and water vapor barrier properties of an edible wheat gluten film. Journal of Food Science, 58, 206e211. He, Q., Zhang, Y., Cai, X., & Wang, S. (2016). Fabrication of gelatineTiO2 nanocomposite film and its structural, antibacterial and physical properties. International Journal of Biological Macromolecules, 84, 153e160. Hosseini, S. F., Rezaei, M., Zandi, M., & Farahmandghavi, F. (2015a). Fabrication of bio-nanocomposite films based on fish gelatin reinforced with chitosan nanoparticles. Food Hydrocolloids, 44, 172e182. Hosseini, S. F., Rezaei, M., Zandi, M., & Farahmandghavi, F. (2015b). Preparation and characterization of chitosan nanoparticles-loaded fish gelatin-based edible film. Journal of Food Process Engineering, 1e9. Hosseini, S. F., Rezaei, M., Zandi, M., & Farahmandghavi, F. (2016). Development of bioactive fish/gelatin chitosan nanoparticles composite films with antimicrobial properties. Food Chemistry, 194, 1266e1274. Ineiguez-Franco, F., Soto-Valdez, H., Peralta, E., Ayala-Zavala, J. F., Auras, R., & GamezMeza, N. (2012). Antioxidant activity and diffusion of catechin and epicatechin from antioxidant active films made of poly(L-lactic acid). Journal of Agricultural and Food Chemistry, 60, 6515e6523. Karbowiak, T., Debeaufort, F., & Voilley, A. (2006). Importance of surface tension characterization for food, pharmaceutical and packaging products: A review. Critical Reviews in Food Science and Nutrition, 46, 391e407. Lemus, M. R. (2011). Models of sorption isotherms for food: Uses and limitations. Vitae, 18, 325e334. Liu, F., Antoniou, J., Li, Y., Majeed, H., Liang, R., Ma, Y., et al. (2016). Chitosan/sulfobutylether-b-cyclodextrin nanoparticles as a potential approach for tea polyphenol encapsulation. Food Hydrocolloids, 57, 291e300. Liu, F., Antoniou, J., Li, Y., Yi, J., Yokoyama, W., Ma, J., et al. (2015). Preparation of gelatin films incorporated with tea polyphenol-nanoparticles for enhancing controlled release antioxidant properties. Journal of Agricultural and Food Chemistry, 63, 3987e3995. Liu, F., Jiang, Y., Du, B., Chai, Z., Jiao, T., Zhang, C., et al. (2013). Design and characterization of controlled-release edible packaging films prepared with synergistic whey-protein polysaccharide complexes. Journal of Agricultural and Food Chemistry, 61, 5824e5833. Liu, F., Majeed, H., Antoniou, J., Li, Y., Ma, Y., Yokoyama, W., et al. (2016b). Tailoring physical properties of transglutaminase-modified gelatin films by varying drying temperature. Food Hydrocolloids, 58, 20e28. Li, K.-K., Yin, S.-W., Yang, X.-Q., Tang, C.-H., & Wei, Z.-H. (2012). Fabrication and characterization of novel antimicrobial films Derived from thymol-loaded zeinesodium caseinate (SC) nanoparticles. Journal of Agricultural and Food Chemistry, 60, 11592e11600. pez-de-Dicastillo, C., Go mez-Estaca, J., Catala , R., Gavara, R., & Herna ndezLo ~ oz, P. (2012). Active antioxidant packaging films: Development and effect Mun on lipid stability of brined sardines. Food Chemistry, 131, 1376e1384.
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pez de Dicastillo, C., Ares Pernas, A., Castro Lopez, M. D. M., Lopez Vilarino, J. M., & Lo Gonzalez Rodriguez, M. V. (2013). Enhancing the release of the antioxidant tocopherol from polypropylene films by incorporating the natural plasticizers lecithin, olive Oil, or sunflower oil. Journal of Agricultural and Food Chemistry, 61, 11848e11857. pez de Dicastillo, C., Nerin, C., Alfaro, P., Catala, R., Gavara, R., & HernandezLo Munoz, P. (2011). Development of new antioxidant active packaging films based on ethylene vinyl alcohol copolymer (EVOH) and green tea extract. Journal of Agricultural and Food Chemistry, 59, 7832e7840. Mahmoud, A. A., El-Feky, G. S., Kamel, R., & Awad, G. E. (2011). Chitosan/sulfobutylether-beta-cyclodextrin nanoparticles as a potential approach for ocular drug delivery. International Journal of Pharmaceutics, 413, 229e236. Mostafavi, F. S., Kadkhodaee, R., Emadzadeh, B., & Koocheki, A. (2016). Preparation and characterization of tragacanth-locust bean gum edible blend films. Carbohydrate Polymers, 139, 20e27. Noronha, C. M., de Carvalho, S. M., Lino, R. C., & Barreto, P. L. (2014). Characterization of antioxidant methylcellulose film incorporated with alpha-tocopherol nanocapsules. Food Chemistry, 159, 529e535. Ooi, S. Y., Ahmad, I., & Amin, M. C. I. M. (2015). Cellulose nanocrystals extracted from rice husks as a reinforcing material in gelatin hydrogels for use in controlled drug delivery systems. Industrial Crops and Products. http://dx.doi.org/10.1016/ j.indcrop.2015.11.082. inz, C., & McHugh, T. H. Otoni, C. G., Avena-Bustillos, R. J., Olsen, C. W., Bilbao-Sa (2016). Mechanical and water barrier properties of isolated soy protein composite edible films as affected by carvacrol and cinnamaldehyde micro and nanoemulsions. Food Hydrocolloids, 57, 72e79. ́ Park, G. S., & Crank, J. (1968). Diffusion in polymers. New York: Academic Press. ́ Ajun, W.́ (2006). Effects of molecular weight, degree of acetylation and Qun, G., & ionic strength on surface tension of chitosan in dilute solution. Carbohydrate Polymers, 64, 29e36. Srinivasa, P. C., Ramesh, M. N., Kumar, K. R., & Tharanathan, R. N. (2003). Properties and sorption studies of chitosanepolyvinyl alcohol blend films. Carbohydrate Polymers, 53, 431e438. Suppakul, P., Chalernsook, B., Ratisuthawat, B., Prapasitthi, S., & Munchukangwan, N. (2013). Empirical modeling of moisture sorption characteristics and mechanical and barrier properties of cassava flour film and their relation to plasticizingeantiplasticizing effects. LWT e Food Science and Technology, 50, 290e297. Teodoro, A. P., Mali, S., Romero, N., & de Carvalho, G. M. (2015). Cassava starch films containing acetylated starch nanoparticles as reinforcement: Physical and mechanical characterization. Carbohydrate Polymers, 126, 9e16. lez-Martínez, C. (2009). Characterization Vargas, M., Albors, A., Chiralt, A., & Gonza of chitosaneoleic acid composite films. Food Hydrocolloids, 23, 536e547. Wan, A., Xu, Q., Sun, Y., & Li, H. (2013). Antioxidant activity of high molecular weight chitosan and N, O-quaternized chitosans. Journal of Agricultural and Food Chemistry, 61, 6921e6928. Wexler, A. S., & Dutcher, C. S. (2013). Statistical mechanics of multilayer sorption: Surface sension. Journal of Physical Chemistry Letters, 4, 1723e1726. Wu, J., Chen, S., Ge, S., Miao, J., Li, J., & Zhang, Q. (2013). Preparation, properties and antioxidant activity of an active film from silver carp (hypophthalmichthys molitrix) skin gelatin incorporated with green tea extract. Food Hydrocolloids, 32, 42e51. Wu, J., Liu, H., Ge, S., Wang, S., Qin, Z., Chen, L., et al. (2015). The preparation, characterization, antimicrobial stability and in vitro release evaluation of fish gelatin films incorporated with cinnamon essential oil nanoliposomes. Food Hydrocolloids, 43, 427e435. Yen, M.-T., Yang, J.-H., & Mau, J.-L. (2008). Antioxidant properties of chitosan from crab shells. Carbohydrate Polymers, 74, 840e844. Yu, S.-H., Tsai, M.-L., Lin, B.-X., Lin, C.-W., & Mi, F.-L. (2015). Tea catechins-crosslinked methylcellulose active films for inhibition of light irradiation and lipid peroxidation induced b-carotene degradation. Food Hydrocolloids, 44, 491e505.
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Food Hydrocolloids journal homepage: www.elsevier.com/locate/foodhyd
Controlled-release of tea polyphenol from gelatin films incorporated with different ratios of free/nanoencapsulated tea polyphenols into fatty food simulants Fei Liu a, b, Roberto J. Avena-Bustillos b, Bor-Sen Chiou b, Yue Li a, Yun Ma a, Tina G. Williams b, Delilah F. Wood b, Tara H. McHugh b, Fang Zhong a, * a
State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, Wuxi, 214122, People's Republic of China Western Regional Research Center, ARS, U.S. Department of Agriculture, Albany, CA, 94710, United States
b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 26 April 2016 Received in revised form 24 July 2016 Accepted 1 August 2016 Available online 3 August 2016
Chitosan nanoparticles with different encapsulation efficiencies were incorporated to obtain gelatin films with controlled-release properties. Nanoparticles tended to aggregate as encapsulation efficiency increased, due to the increase in surface tension of film-forming solutions. The addition of nanoparticles increased the compactness and isotherm hysteresis of films, whereas decreased the moisture diffusion. Tea polyphenol released faster and greater from films in 50% ethanol (4 C) than in 95% ethanol (25 C) fatty food stimulant due to the swelling by water. However, controlled-release showed in both simulants, where free tea polyphenol had the largest release followed by 51.3%, 83.3% and 96.9% encapsulation efficiency. The diffusion coefficient in 50% ethanol was 2 orders of magnitude higher than that 95% ethanol (1013 and 1011 cm2/s, respectively). Films maintained their structures in 95% ethanol after 240 h but not in 50% ethanol. These results might open up new designs in long-term protection for fatty foods. © 2016 Elsevier Ltd. All rights reserved.
Keywords: Tea polyphenol Gelatin film Chitosan nanoparticle Controlled-release Fatty food simulant Antioxidant Chemical compounds studied in this article: Chitosan hydrochloride (PubChem CID: 71853) Sulfobutylether-b-cyclodextrin sodium (PubChem CID: 66577045)
1. Introduction Synthetic antioxidants are being substituted by natural antioxidants due to their potential toxicity and carcinogenicity. Tea polyphenol (TP), a mixture of catechins from tea, can inhibit the pez de Dicastillo et al., 2011; Noronha, oxidation of food systems (Lo de Carvalho, Lino, & Barreto, 2014). However, the hydrophilicity restricts their application in oils and the direct addition might cause pez de Dicastillo, unfavorable reactions with food components (Lo Ares Pernas, Castro Lopez, Lopez Vilarino, & Gonzalez Rodriguez, 2013; Liu et al., 2015). Active antioxidant film is a novel alternative to overcome these limitations. They can also provide
* Corresponding author. Tel.: þ86 510 85197876; fax: þ86 510 85197876. E-mail address: [email protected] (F. Zhong). http://dx.doi.org/10.1016/j.foodhyd.2016.08.004 0268-005X/© 2016 Elsevier Ltd. All rights reserved.
continuous release to the food surface where oxidation occurs most lu, 2014). intensively (Arcan & Yemeniciog More recently, the demand for prolonging the shelf life of food has encouraged the development of controlled-release films, where antioxidants release in a slow, but controlled manner, to maintain food quality for a longer time (Chen, Lee, Zhu, & Yam, 2012). The inclusion of antioxidants in the matrix of nanoparticles might be a valid method to control the release process due to the increase in mez-Estaca, Lo pez-de-Dicastillo, tortuosity of diffusion paths (Go ndez-Mun ~ oz, Catal Herna a, & Gavara, 2014). The controlledrelease of active films have been obtained by incorporating nanoparticles, such as zeinsodium caseinate nanoparticles (Li, Yin, Yang, Tang, & Wei, 2012), lecithin nanoliposomes (Wu et al., 2015), and poly ε-caprolactone nanocapsules (Noronha et al., 2014). Chitosan nanoparticles are a potential alternative to encapsulate TP and provide controlled-release properties for films. Also,
F. Liu et al. / Food Hydrocolloids 62 (2017) 212e221
213
chitosan nanoparticles can improve the thermal mechanical properties of films (Hosseini, Rezaei, Zandi, & Farahmandghavi, 2015a, 2016). In our previous study, TP-loaded chitosan nanoparticles, prepared by the ionotropic gelation between chitosan hydrochloride (CSH) and sulfobutyl ether-b-cyclodextrin sodium (SBE-b-CD), have been successfully incorporated into gelatin films (Liu et al., 2015). The protection effects for sunflower oil within the bags made of films varied with the encapsulation efficiency (EE) of TP in CSH-SBE-b-CD nanoparticles (CSNs). However, the causes of the differences in protection effects were still not clearly know. The differences in release properties of TP from gelatin films were not directly investigated and compared. The release of antioxidants from films is a complex process relating to the polymer matrix, pez-de-Dicastillo, nature of antioxidants and properties of foods (Lo mez-Estaca, Catala , Gavara, & Hern ~ oz, 2012). Go andez-Mun The objective of this study was to compare the release properties of TP from gelatin films, with different ratios of free TP to nanoencapsulated TP, into fatty food simulants. We wanted to confirm if the controlled-release of TP could be obtained by varying the EE of TP without the increase of content, where the free TP would release faster while the encapsulated TP release slower to extend the protection effect. CSNs with different EEs were prepared by adjusting the concentration of CSH. The dispersion of CSNs within the gelatin matrix was examined to determine its influence on the final properties and applications of films (Teodoro, Mali, Romero, & de Carvalho, 2015). The films’ antioxidant properties were investigated by monitoring the release of TP into two different fatty food simulants and their DPPH radical scavenging activity. The physical, morphological and structural properties of films were monitored as well.
hydrating gelatin powders in distilled water for 1 h at room temperature and then heating to 65 C with continuous stirring. Glycerol (20% w/w of gelatin) was added as a plasticizer to reduce the brittleness of films. The gelatin solution (25 mL) was then added to 75 mL of freshly obtained nanoparticle suspension or 75 mL of water/TP-containing aqueous solution (0.25 mg/mL). After stirring for 1 h, 35 mL of the film forming solution was cast in a square Petri dish (10 cm 10 cm) and then dried at room temperature (23 C) in darkness for 2 days until it reached a constant weight. Films were conditioned at 25 C and 53% relative humidity (RH) for at least 48 h before testing.
2. Materials and methods
2.4. Moisture sorption isotherm of films
2.1. Materials
Water vapor adsorption-desorption isotherms were obtained at 25 C on a dynamic vapor sorption (DVS) Advantage apparatus (Surface Measurement Systems NA, Allentown, Pa., USA). Prior to analysis, film samples were dehydrated in a desiccator at 25 C until reaching equilibrium. About 5 mg of the film was loaded in a weighing basket and the change in mass was recorded over a humidity range of 0e98%. The diffusion coefficients were calculated from the adsorption-desorption kinetics according to Park and Crank (1968) using the DVS Analysis software (Version 6.3, Surface Measurement Systems Ltd, UK). The Brunauer, Emmett and Teller (BET) surface area was calculated from the linear part of the BET plot according to IUPAC recommendations.
Gelatin (type B from bovine skin, ~200 Bloom value) and glycerol were purchased from China Medicine (Group) Shanghai Chemical Reagent Co. (Shanghai, China). Chitosan hydrochloride (CSH), with a molecular weight of 100 kDa, a degree of deacetylation of 86% and derived from crab shells, was obtained from Golden-Shell Biochemical Co., Ltd. (Hangzhou, China). Sulfobutyl ether-b-cyclodextrin sodium (SBE-b-CD, food grade), with a molecular weight of 2.082 kDa and an average degree of substitution of 6, was purchased from Kunshan Chemical Industries Co., Ltd. (Jiangsu, China). Tea polyphenol (TP), with a polyphenol content 98% and catechins content 90% (determined by manufacturer), was obtained from Qiangsheng Medicine Science Technology Co., Ltd. (Shanghai, China). 2,2-Diphenyl-1-picrylhydrazyl (DPPH) and gallic acid was purchased from Sigma Chemical Co. (St. Louis, MO, USA). All other reagents were analytical grade. 2.2. Preparation of films Gelatin films embedded with various ratios of free to encapsulated TP were fabricated by incorporating CSNs with three different TP EEs (51.3%, 83.3% and 96.9%) according to our previous studies (Liu, Antoniou, et al., 2016; Liu et al., 2015). First, 1 mg/mL TP was added to 24 mL of 2, 4 or 8 mg/mL SBE-b-CD and the solutions were mildly stirred at room temperature for 24 h to promote the formation of inclusion complexes. These solutions were then added drop-wise to 72 mL of 0.5, 1 or 2 mg/mL CSH solutions with vigorous magnetic stirring, respectively. The blank CSNs without TP were also prepared as controls. Freshly obtained CSNs were immediately used to prepare films. Subsequently, 8% (w/v) gelatin solutions were prepared by
2.3. Characterization of film-forming solutions 2.3.1. Scanning electron microscopy (SEM) analysis To observe the dispersion of CSNs within the gelatin matrix, one drop (10 mL) of CSN suspensions and film-forming solutions was placed on the surface of aluminum foil, dried at room temperature and then sputter coated with gold under vacuum. SEM images were obtained using a Hitachi S-4700 scanning electron microscope (Hitachi, Japan) operating at 5 kV. 2.3.2. Surface tension measurement The surface tension of CSN suspensions and film-forming solutions was determined with a Krüss K100 Force Tensiometer (Germany) at room temperature according to Mostafavi, Kadkhodaee, Emadzadeh, and Koocheki (2016) Ultrapure water was used in the calibration before the measurement. The Wilhelmy plate method was used and surface tension was recorded for 120 s after placing the platinum plate on the dispersion surface.
2.5. Release study of films Two food simulants, 95% ethanol and 50% ethanol, were used to perform the release studies. The 95% ethanol can be regarded as a simulant for fats, oil and fatty foods due to their similar hydrophobicity (Noronha et al., 2014). The 50% ethanol can be regarded as a simulant for oil in water emulsions and alcoholic beverages (Ineiguez-Franco et al., 2012). The release of tea polyphenols (TP) from films was determined by adding film pieces (3 3 cm) and 30 mL of food simulants into 50 mL glass vials with screw caps containing a PTFE/silicon septum. Release tests were conducted in the dark at 25 C for 95% and 4 C for 50% ethanol simulants, respectively. The food simulant (0.1 mL) was collected and its TP content was determined at predetermined times (0, 1, 2, 4, 6, 8, 10, 24, 48, 72, 96, 144, 168, 192, 216, 240 h). Simulants were deoxygenated with bubbling nitrogen and the vials were flushed with nitrogen before closing. All analyses were measured in triplicate with three different vials. The release kinetics when the released TP amount was lower than 0.6 of initial TP
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loading were calculated according to:
Mt 4 Dt ¼ Lp p Mp
1=2 (1)
where Mt was the mass of TP in the food simulants at a particular time t (s), MP was the initial TP loading in the gelatin films, Lp was film thickness and D was the diffusion coefficient. D was determined from the slope of the plot of Mt/Mp as a function of t1/2 (Arrieta et al., 2014; Chen et al., 2012).
2.5.1. Total phenol assay The amount of TP released from films was measured by using the Folin-Ciocalteu method (Yu, Tsai, Lin, Lin, & Mi, 2015). Food simulant (20 mL) was thoroughly mixed with 100 mL of FolinCiocalteu reagent (10% v/v). Then, 80 mL of 7.5% (w/v) sodium carbonate was added to the mixture and the sample was allowed to stand for 60 min at room temperature. The absorbance was measured at 725 nm in a Microplate Reader (Shimadzu UV-2450, Japan). Total phenol content was expressed as mg gallic acid equivalent/g film sample according to:
Fig. 1. SEM micrographs of selected CSN suspensions (magnification: 1000 and 10,000) and film-forming solutions (magnification: 10,000) dried on the surface of aluminum foil. xCSH-TP, TP-loaded CSNs prepared at the indicated CSH concentrations. Gel-TP and Gel-xCSH-TP, films prepared with gelatin þ TP-loaded CSNs at the indicated CSH concentrations (mg/mL).
F. Liu et al. / Food Hydrocolloids 62 (2017) 212e221
Total Phenol Content ¼ ðC V Þ=M
(2)
where C was the concentration of gallic acid obtained from the standard curve (mg/mL), V was the volume of the food simulant (mL) and M was the weight of added film (mg). 2.5.2. DPPH radical scavenging activity A previously described method was used to evaluate the DPPH radical scavenging activity (Liu et al., 2015), with slight modifications. Food simulant (80 mL) was mixed with 100 mL (0.1 mM) of DPPH dissolved in ethanol. The mixture was kept in the dark for 30 min at room temperature. The absorbance was measured at 517 nm in a Microplate Reader (Shimadzu UV-2450, Japan). The assay was carried out in triplicate. The DPPH radical scavenging activity was determined according to:
DPPH scavenging ð%Þ ¼ 100 ð1 ðAs Ab Þ=Ac
(3)
where As was the sample absorbance after addition of food simulant, Ab was the absorbance of the food simulant and Ac was the absorbance of the control sample containing blank food simulant mixed with the DPPH solution. 2.6. Film characterization during release study 2.6.1. Swelling tests Swelling degree (SD) of films was monitored during release studies in the food simulants according to the method of Yu et al. (2015). Film samples (3 3 cm) were previously dried and weighted before immersion into 30 mL of the food simulants. At selected times (0, 2, 10, 24, 48, 72, 96, 144, 168, 192, 240 h), film samples were removed from the glass vials and carefully wiped of excess liquid. The weight was measured and the film samples were returned to the vials. SD was calculated from the initial weight of dried film samples (m0) and the weight of swollen films at each time (mt), using:
SD ð%Þ ¼ 100 ð1 þ ðmt m0 Þ=m0 Þ
(4)
2.6.2. Scanning electron microscopy (SEM) and X-ray diffraction (XRD) Film samples were taken out from the glass vials and dried at ambient temperature after 0, 24, 96, 240 h exposure to the food simulants. The cross sections of films were characterized with a scanning electron microscope (SEM, S-4800, Hitachi, Japan) under high vacuum mode at an accelerating voltage at of 2 kV. Film pieces were cryofractured after freezing in liquid nitrogen to obtain the cross sections. Samples were mounted on the specimen holder using double-sided adhesive tapes and then gold coated with a sputter coater (Emitech K550X, Quorum Technologies Inc., UK) under 15 mA for 45 s. The changes in crystalline phases of gelatin films during release tests were determined by XRD. XRD patterns of films were obtained by an X-ray diffractometer (D8 Phaser, Bruker AXS Germany) with Cu Ka radiation (1.542 Å) operating at 40 kV and 40 mA. Data were recorded in the range (2q) of 3e60 with a step size of 0.02 and a scanning rate of 4 min1.
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test was used to determine the significant differences of the mean values (P < 0.05). 3. Results and discussion 3.1. Dispersion of CSNs within film matrix As shown in Fig. 1, individual nanoparticles could be identified in CSN suspensions at low CSH concentration (0.5 mg/mL). However, particles became aggregated with an increase in CSH concentration and the spherical shape was also changed at 2 mg/mL of CSH. This aggregation might be due to the enhanced hydrogen bonding interactions between CSNs at higher concentrations during the drying process (Hosseini, Rezaei, Zandi, & Farahmandghavi, 2015b). A smooth and homogeneous surface morphology was observed for the control film (Gel-TP). The incorporation of CSNs into the film-forming solution led to the development of a rougher surface. At low CSH concentration (0.5 mg/mL), CSNs appeared to be dispersed evenly within the gelatin matrix. Also, the agglomeration between CSNs in the films occurred with an increase in CSH concentration. CSNs collided and aggregated in the gelatin matrix during drying and the frequency of collision increased at higher CSH concentrations (Hosseini et al., 2016; Wu et al., 2015). However, a denser and more compact surface structure was obtained at higher CSH concentrations due to strong bonding between CSNs and gelatin. Similar results were also reported by Hosseini et al. (2015a) in the fabrication of chitosan nanoparticle composite gelatin films. 3.2. Surface tension of CSN suspensions and film-forming solutions Surface tension can be used to better understand the formulation and processing of edible films or coatings, including interactions between components, wetting or encapsulation (Karbowiak, Debeaufort, & Voilley, 2006). Samples with gelatin or higher CSN concentrations took longer to reach their equilibrium surface tension value. This is shown in the plot of surface tension as a function of time in Fig. S1. The presence of CSNs caused a decrease in surface tension values (P < 0.05) in both CSN suspensions and film-forming solutions (Fig. 2). This could be attributed to the lower surface tension of chitosan solution (61.8 mN/m) compared to that lezof TP solution (71.4 mN/m) (Vargas, Albors, Chiralt, & Gonza
2.7. Statistical analysis Data were presented as mean value ± standard deviation. The data were analyzed by one-way analysis of variance (ANOVA) using the SPSS 19.0 package (IBM, New York). Duncan's multiple range
Fig. 2. Surface tension of CSN solutions and related film-forming solutions. TP and xCSH-TP, TP/CSN suspensions and corresponding film-formation solutions prepared at the indicated CSH concentrations (mg/mL).
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Martínez, 2009). More CSNs absorbed on the surface of CSN suspensions at higher CSH concentrations, leading to the further decrease in surface tension. The 0.5 mg/mL CSH film-forming solution had the lowest
surface tension. The addition of gelatin could change the CSH structure and distribution of CSNs within the solutions due to interactions between CSNs and gelatin (Mostafavi et al., 2016). Furthermore, glycerol tended to reduce the surface tension of filmforming solutions (Caro et al., 2016; Wexler & Dutcher, 2013). An optimum dispersion of CSNs in the film-forming solutions as well as the lowest intermolecular forces between CSNs, gelatin and glycerol might have led to the lowest surface tension for the 0.5 mg/ mL CSH sample (Qun & Ajun, 2006). According to Mostafavi et al. (2016), the suspension spreadability and coating integrity critically depended on the surface tension of solutions. Therefore, higher surface tensions induced by the increased CSH concentrations might have led to the aggregation of CSNs and the formation of beads within the gelatin matrix (Fig. 1). 3.3. Moisture sorption isotherm and diffusion coefficient Equilibrium moisture contents of films varied with the water vapor pressure surrounding them. The adsorption and desorption isotherms of all films had a sigmoidal shape (Fig. 3a, b), showing an asymptotic trend as water activity (aw) approached 1 (Suppakul, Chalernsook, Ratisuthawat, Prapasitthi, & Munchukangwan, 2013). The incorporation of CSNs resulted in films with lower equilibrium moisture contents, especially at aw > 0.6. Similar
Fig. 3. Moisture (a) adsorption and (b) desorption isotherms of gelatin films. (c) Hysteresis values of adsorption and desorption isotherms. Gel-TP and Gel-xCSH-TP, films prepared with gelatin þ TP-loaded CSNs at the indicated CSH concentrations (mg/mL).
Fig. 4. Diffusion coefficients of (a) adsorption and (b) desorption process of gelatin films as a function of target relative humidity. Gel-TP and Gel-xCSH-TP, films prepared with gelatin þ TP-loaded CSNs at the indicated CSH concentrations (mg/mL).
F. Liu et al. / Food Hydrocolloids 62 (2017) 212e221
results were obtained by Teodoro et al. (2015) after incorporating acetylated starch nanoparticles into cassava starch films. The addition of CSNs resulted in CSN-gelatin interactions, leading to a reduced number of polar sites available for hydrogen bonding with water molecules (Erbas, Aykin, Arslan, & Durak, 2016). The hysteresis in films, which was related to the nature and state of components (Lemus M, 2011), also reflected the rearrangements of structure and conformation of gelatin in the presence of CSNs (Fig. 3c). The largest hysteresis corresponded to the lowest relaxation rate in the adsorption/desorption process and was observed for the Gel-1CSH-TP sample (Gontard, Guilbert, & Cuq, 1993), which had the most compact microstructure. As shown in Fig. 4, good agreement was observed between the diffusion coefficients and the sorption isotherms. The addition of CSNs resulted in a decrease in the diffusion coefficient values, with the lowest diffusion coefficient obtained for the Gel-1CSH-TP sample in the relative humidity (RH) range of 30e60%., The moisture diffusion coefficient was also dependent on RH, similar to the inz, and McHugh results of Otoni, Avena-Bustillos, Olsen, Bilbao-Sa (2016). The diffusion of moisture occurred by several mechanisms, including adsorption to the monolayer, vapor transport through a swollen matrix and collapse of the structure (Otoni et al., 2016). Moreover, the barrier property of films depended on the moisture diffusion coefficient and its transport within the matrix (Srinivasa, Ramesh, Kumar, & Tharanathan, 2003). Therefore, the diffusion coefficient values could be used to explain the differences in water barrier properties of films. These values correlated well with the WVP data (Table S1). In addition, the volume of vapor monolayer (Vm) and the corresponding specific surface area of gelatin films could be calculated from the adsorption isotherm (aw < 0.45) using the classical BET equation (Table 1). The changes in these values
Table 1 Vm and Specific Surface Area of Gelatin Films Calculated from the BET Model when aw < 0.45.a Films
Vm (mmol/g)
Gel-TP Gel-0.5CSH-TP Gel-1CSH-TP Gel-2CSH-TP
3.3 2.6 2.1 2.9
± ± ± ±
0.1a 0.1c 0.1d 0.1b
Specific surface area (m2/g) 229.1 181.1 143.9 199.2
± ± ± ±
6.9a 5.4c 4.3d 6.0b
Different lowercase letters in the same column indicate a statistically significant difference (P < 0.05). Gel-TP and Gel-xCSH-TP, films prepared with gelatin þ TPloaded CSNs at the indicated CSH concentrations (mg/mL). Vm, the amount of vapor adsorbed if a monolayer was to form. a Values are expressed as mean ± standard deviation.
217
were consistent with the sorption isotherm and diffusion coefficient results. The differences in the specific surface areas might have affected the release of TP from gelatin films. 3.4. Swelling degree (SD) Food simulant resistance and release of active compounds were related to the swelling of films (Garde, Catala, Gavara, & Hernandez, 2001; Yu et al., 2015). Gelatin is a hydrophilic polymer that can swell in contact with water, which would facilitate the diffusion of active compound through its matrix (Buonocore, Del Nobile, Panizza, Corbo, & Nicolais, 2003). Different swelling degrees were observed when the films were placed in 95% and 50% ethanol fatty food simulants (Fig. 5). The gelatin films immersed in 95% ethanol swelled slowly, whereas the films immersed in 50% ethanol swelled more quickly. In 95% ethanol, the films containing CSNs had slightly lower SD. Also, an increase in CSH concentration resulted in a further decrease in SD (Fig. 5a). These results could be due to an increase in intermolecular attractive forces between CSNs and gelatin at higher CSH concentrations, resulting in reduced free volume and a more compact and less permeable film matrix (Hosseini et al., 2015a). In comparison, the addition of CSNs to films led to higher SD in 50% ethanol (Fig. 5b). This might be attributed to the slight hydrophilic nature of CSNs as reported by Ooi, Ahmad, and Amin (2015). The reduced free volume in films with the highest CSH concentrations (2 mg/mL) also restricted the gelatin chain movements and reduced the swelling capability of films in 50% ethanol. 3.5. Release of TP from films into fatty food simulants The release of TP from gelatin films into 50% ethanol fatty food simulants was measured at 4 C, whereas the release into 95% ethanol was carried out at 25 C to monitor the TP release (Fig. 6). In each simulant, all films had similar release profiles with an initial fast release followed by a sustained slow release. The initial fast release of TP provided the short-term protection and the subsequent slower release guaranteed the intermediate/long-term protection (Chen et al., 2012). The total TP release after 240 h could be controlled by the amount of CSNs added to the films. For example, the Gel-2CSH-TP film in both simulants released approximately half of the TP released from the blank films (Gel-TP). Also, the extent of TP release depended on the amount of free TP within the gelatin matrix, which was related to the encapsulation efficiency (EE). The EE could be controlled by the CSH concentration used for CSN
Fig. 5. Swelling degree (SD) of gelatin films immersed into (a) 95% ethanol (25 C) and (b) 50% ethanol (4 C) fatty food simulants as a function of time. Gel-TP and Gel-xCSH-TP: films prepared with gelatin þ TP-loaded CSNs at the indicated CSH concentrations (mg/mL).
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Fig. 6. Release of TP from gelatin films (left) and related DPPH radical scavenging activity of fatty food simulants (right) of (a) 95% ethanol (25 C) and (b) 50% ethanol (4 C) fatty food simulants as a function of time. Inset is the straight line representation of Mt/Mp vs. t1/2 according to equation (1). Gel-TP and Gel-xCSH-TP: films prepared with gelatin þ TPloaded CSNs at the indicated CSH concentrations (mg/mL).
Table 2 Diffusion Coefficient (D) of TP Released from Gelatin Films into 95% (25 C) and 50% Ethanol (4 C) Fatty Food Simulants.a Films
Gel-TP Gel-0.5CSH-TP Gel-1CSH-TP Gel-2CSH-TP
95% Ethanol
50% Ethanol
D 1013 (cm2/s)
R2
D 1011 (cm2/s)
R2
1.59 1.46 0.58 0.38
0.987 0.988 0.985 0.978
5.54 1.19 2.04 0.39
0.969 0.948 0.9303 0.9646
a Gel-TP and Gel-xCSH-TP, films prepared with gelatin þ TP-loaded CSNs at the indicated CSH concentrations (mg/mL).
preparation, which decreased with increasing CSH concentration, according to our previous studies (Liu, Antoniou, et al., 2016; Liu et al., 2015). As shown in the inset graphs of Fig. 6, the model (1) fitted the experimental data well (R2 > 0.9) and the D values are shown in Table 2. The samples in 50% ethanol had D values that were 2 orders of magnitude higher than those in 95% ethanol. The intake of more water in 50% ethanol (see the SD of films in Fig. 5) led to the plasticization and an increase in D values (Buonocore et al., 2003; pez-de-Dicastillo et al., 2012). The addition of CSNs led to a Lo slower release of TP, with the 2 mg/mL CSH sample having the
Fig. 7. The intensity ratio of crystal and amorphous peak (It/Ia) of gelatin films exposed to (a) 95% ethanol (25 C) and (b) 50% ethanol (4 C) fatty food simulants at the selected release times. Gel and Gel-xCSH, films prepared with gelatin þ control or TP-loaded CSNs at the indicated CSH concentrations (mg/mL).
F. Liu et al. / Food Hydrocolloids 62 (2017) 212e221
219
Fig. 8. SEM micrographs (magnification 10,000) of cross section of gelatin films exposed to (a) 95% ethanol (25 C) and (b) 50% ethanol (4 C) fatty food simulants at the selected times (h). Gel-TP and Gel-xCSH-TP, films prepared with gelatin þ TP-loaded CSNs at the indicated CSH concentrations (mg/mL).
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slowest release. The D values decreased from 1.59 1013 to 0.38 1013 cm2/s and from 5.54 1011 to 0.39 1011 cm2/s in 95% ethanol and 50% ethanol, respectively. The presence of CSNs increased the tortuosity of the TP diffusion path and modified the compactness of the gelatin film structure, thus hindering the mez-Estaca et al., 2014; Liu et al., 2013). The release release of TP (Go of TP from Gel-1CSH-TP film was faster than that from Gel-0.5CSHTP film in 50% ethanol (Fig. 6b), which is different from that in 95% ethanol fatty food simulants. This difference might be due to the higher SD of Gel-1CSH-TP film in 50% ethanol (Fig. 5b). In addition to the diffusion of TP from film, the release in 50% ethanol also resulted from the diffusion of simulant into film and the relaxation of film matrix (Buonocore et al., 2003). pez-de-Dicastillo et al. (2012), it was necessary According to Lo to verify that the active agents retained their antioxidant activity during the release. DPPH radical scavenging activity was used to determine the antioxidant activity of the samples. As shown in Fig. 6 (right), the DPPH radical scavenging activity was proportional to the released TP concentration in 95% ethanol during all the release times. In comparison, this relationship was only observed before 8 h of release in the 50% ethanol. The differences in DPPH radical scavenging activity between various gelatin films were negligible (P > 0.05) after 8 h of release. This similarity could be due to the presence of radical scavenging gelatin and chitosan once they dissolved in the 50% ethanol (Liu et al., 2015; Wan, Xu, Sun, & Li, 2013; Yen, Yang, & Mau, 2008). 3.6. Changes in gelatin film structure during release study All gelatin films had an XRD pattern characteristic of a partially crystalline material, with a sharp peak at ~7 (2q) and a broad peak at ~20 (2q), which corresponded to the triple-helical crystalline structure and the amorphous fractions, respectively (Fig. S2) (Ge et al., 2015; He, Zhang, Cai, & Wang, 2016; Liu, Majeed, et al., 2016). The XRD patterns of gelatin films with blank CSNs (without TP) were also determined as controls. The presence of TP decreased the intensity of the triple-helix peaks. Also, the CSH and SBE-b-CD peaks in the XRD patterns disappeared after forming CSNs, according to Mahmoud, El-Feky, Kamel, and Awad (2011). Therefore, the decrease in the triple-helix peaks might be due to hydrogen bonding between gelatin and TP (Wu et al., 2013), which inhibited the triple-helix formation. The relative triple-helix content could be expressed as the intensity ratio of crystal to amorphous peaks (It/Ia), as shown in Fig. 7. The incorporation of nanoparticles destroyed the hydrogen bonds between gelatin molecules (He et al., 2016), resulting in a decrease in triple-helix content at higher CSH concentrations. Arrieta et al. (2014) reported that an increase in the amount of crystalline regions resulted in slower diffusion of catechin from poly (lactic acid)-poly (hydroxybutyrate) blend films. However, the opposite trend occurred in our study, with samples of higher crystallinity having faster release rates (Fig. 6, Table 2). Although the CSNs retarded some formation of triple helices, the release of TP was mainly controlled by its diffusion from CSNs. Also, all gelatin films that remained in the simulants for longer times had lower triplehelix contents. This decrease in triple-helix content might be due to the modification and relaxation of gelatin conformation as food simulants diffused into the matrix. Furthermore, some tightly bound water, which was necessary to form and stabilize the gelatin triple-helix (Frazier & Srubar, 2016), might have been displaced by ethanol. The microstructure changes of gelatin films during TP release were examined by SEM. In contrast to the film-forming solutions dried on the aluminum foil surface (Fig. 1), the films had smooth surfaces even at the highest CSH concentration (Fig. S3, 2 mg/mL).
This might be explained by the uniform dispersion of CSNs within the gelatin matrix (Fig. 8). Although samples with higher CSH concentrations showed rougher surfaces, no individual CSNs could be identified in their cross-section micrographs. According to Arrieta et al. (2014), the integrity of films, especially those made of biodegradable polymers, should be maintained during the shelf life of the food. As shown in Fig. 8, all gelatin films became rougher at longer release times, which might be due to the plasticization effect pez-de-Dicastillo et al., 2012). Films in (Buonocore et al., 2003; Lo 50% ethanol had rougher surfaces than those in 95% ethanol. Also, the greater penetration of the 50% ethanol solution into gelatin films (Fig. 5) led to the presence of voids or wrinkles in the surface micrographs (Fig. S3b). Therefore, gelatin films containing CSNs were more suitable for use in oils rather than in oil in water emulsions since their microstructures could be better maintained. 4. Conclusions In conclusion, desirable controlled-release properties of gelatin films were obtained by incorporating different ratios of free to nanoencapsulated TP. The release of TP from films was fatty food simulant- and EE-dependent. The release process was related to the morphological changes. The lower the ethanol ratio, the higher in TP release. The higher release process was accompanied with the loss in film integrity. The higher the EE, the lower the ratio of free to encapsulated TP, and thus, the lower the ultimate release percentage. The presence of CSNs increased the tortuosity of TP diffusion paths into simulants, which further increased by increasing CSH concentration in CSNs preparation. Therefore, the free TP released at a faster rate could maintain the short-term antioxidant properties for fatty foods, whereas the encapsulated TP released at a slower rate could maintain the intermediate and/or long-term antioxidant properties. Acknowledgements This work was financially supported by the National 125 Program 2011BAD23B02, 2013AA102207, National Natural Science Foundation of China 31171686, 31401532 and 31571891, Natural Science Foundation of Jiangsu Province BK2012556, 111 Project B07029, PCSIRT0627, JUSRP11422 and JUSRP51507 and the Selfdetermined Research Program of Jiangnan University, JUSRP 115A22. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.foodhyd.2016.08.004. References lu, A. (2014). Controlled release properties of zeinfatty acid Arcan, I., & Yemeniciog blend films for multiple bioactive compounds. Journal of Agricultural and Food Chemistry, 62, 8238e8246. Arrieta, M. P., Castro-Lopez Mdel, M., Rayon, E., Barral-Losada, L. F., LopezVilarino, J. M., Lopez, J., et al. (2014). Plasticized poly(lactic acid)poly(hydroxybutyrate) (PLA-PHB) blends incorporated with catechin intended for active food-packaging applications. Journal of Agricultural and Food Chemistry, 62, 10170e10180. Buonocore, G. G., Del Nobile, M. A., Panizza, A., Corbo, M. R., & Nicolais, L. (2003). A general approach to describe the antimicrobial agent release from highly swellable films intended for food packaging applications. Journal of Controlled Release, 90, 97e107. pez, L., Abugoch, L., & Tapia, C. (2016). Novel Caro, N., Medina, E., Díaz-Dosque, M., Lo active packaging based on films of chitosan and chitosan/quinoa protein printed with chitosan-tripolyphosphate-thymol nanoparticles via thermal ink-jet printing. Food Hydrocolloids, 52, 520e532. Chen, X., Lee, D. S., Zhu, X., & Yam, K. L. (2012). Release kinetics of tocopherol and quercetin from binary antioxidant controlled-release packaging films. Journal of
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