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bacterial cellulose nanofiber composite films
Accepted Manuscript Title: Drug release and antioxidant/antibacterial activities of silymarin-zein nanoparticle/bacterial cellulose nanofiber composite films Authors: Yi-Hsuan Tsai, Yu-Ning Yang, Yi-Cheng Ho, Min-Lang Tsai, Fwu-Long Mi PII: DOI: Reference:
S0144-8617(17)31142-6 https://doi.org/10.1016/j.carbpol.2017.09.100 CARP 12845
To appear in: Received date: Revised date: Accepted date:
8-8-2017 28-9-2017 29-9-2017
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Drug release and antioxidant/antibacterial activities of silymarin-zein nanoparticle/bacterial cellulose nanofiber composite films
Yi-Hsuan Tsai1, Yu-Ning Yang1, Yi-Cheng Ho2, Min-Lang Tsai1*, Fwu-Long Mi3,4,5*
1. Department of Food Science, National Taiwan Ocean University, Keelung 20224, Taiwan, ROC 2. Department of Bioagriculture Science, National Chiayi University, Chiayi 60004, Taiwan. 3. Department of Biochemistry and Molecular Cell Biology, School of Medicine, College of Medicine, Taipei Medical University, Taipei 11031, Taiwan, ROC 4. Graduate Institute of Medical Sciences, College of Medicine, Taipei Medical University, Taipei 110, Taiwan 5. Graduate Institute of Nanomedicine and Medical Engineering, College of Biomedical Engineering, Taipei Medical University, Taipei 11031, Taiwan
Correspondence to: Fwu-Long Mi, PhD Professor Department of Biochemistry School of Medicine College of Medicine Taipei Medical University Taipei, Taiwan 110, ROC Fax: 886-2-2735-6689 E-mail: [email protected] The two corresponding authors (Min-Lang Tsai1* and Fwu-Long Mi2,5*) contributed equally to this work
1
ABSTRACT Bacterial cellulose (BC) is a biopolymer composed of nanofibers which has excellent film-forming ability. However, BC do not have antibacterial or antioxidant activity, thus limiting the applicability of BC for food and biomedical applications. In this study, flavonoid silymarin (SMN) and zein were assembled into spherical SMN-Zein nanoparticles that could be effectively adsorbed onto BC nanofibers. SMN-Zein nanoparticles greatly changed the wettability
and
swelling
property
of
BC
films
due
to
the
formation
of
nanoparticles/nanofibers nanocomposites. SMN-Zein nanoparticles ehanced the release of sparingly soluble silymarin from the nanocomposite films. The active films showed more effective antioxidant and antibacterial activities as compared with pure BC films and thus were able to protect salmon muscle from deterioration and lipid oxidation. These findings suggest that the nanoparticle/nanofiber composites may offer a suitable platform for modification of BC films with improved drug release properties and biological activities. Keywords: bacterial cellulose, zein, drug release, active films, antibacterial, antioxidant
2
1. Introduction Bacterial cellulose (BC) is a biopolymer that can be synthesized by Acetobacter xylinum, which receives growing interest due to its higher biocompatibility, water holding capacity and porosity. BC has become a promising material for biomedical applications, including drug delivery, wound healing and tissue regeneration (Abeer, Amin, & Martin, 2014). BC has excellent film-forming and emulsion-stabilizing properties, making it suitable for use in food applications, such as gelling agents, food stabilizer and food packaging materials. Lipid oxidation and microbiological spoilage are the major causes of quality deterioration in food products. Unfortunately, BC films do not have any antibacterial or antioxidant activity, thus it should be incorporated with active ingredients for food preservation or biomedical purposes. BC films incorporating antioxidants or antimicrobials such as, benzoic acid derivatives, tetracycline and ε-polylysine have been developed to overcome the obstacles (Gao, Zhang, Song, & Hou, 2014; Shao et al., 2016; Sukhtezari, Almasi, Pirsa, Zandi, & Pirouzifard, 2017). Flavonoids is one of the most abundant naturally occurring products can inhibit the growth of microorganisms and protect cells from oxidative stress. Silymarin, a naturally occurring flavonoid isolated from Silybum marianum (L.) Gaertn. (milk thistle), has antioxidant activity against lipid peroxidation (Razavi-Azarkhiavi et al., 2014). Silymarin also demonstrated antimicrobial activity against pathogenic fungi, viruses and bacteria (Yun, & Lee, 2017; Lee, et al., 2003) and exhibited antiadherent/antibiofilm activity against S. epidermidis (Evren, & Yurtcu, 2015). However, silymarin is poorly soluble in water, causing the low availability of silymarin. Various methods have been developed with the aim to enhance aqueous solubility and bioavailability of silymarin, such as microemulsion, proliposome, poly(ethylene glycol) 400 (PEG 400) and phospholipid-based complexes (Theodosiou, Purchartova, Stamatis, Kolisis, & Kren, 2014). 3
In the development of a BC-based composite film, scaffold, wound dressing or food packaging, it is a challenge to incorporate a water-insoluble active compound into the BC matrix and then controlled the release of this active ingredient. Antibacterial BC composites were developed by the incorporation of silver or zinc oxide nanoparticles (Janpetch, Saito, & Rujiravanit, 2016; Jebel, & Almasi, 2016), yet these BC-based nanocomposites can’t release active compounds to exert antioxidant and anti-inflammatory effects. To overcome the problems, we developed a new type of antioxidant and antibacterial BC nanocomposites by incorporating
BC
films
with
silymarin-zein
(SMN-Zein)
nanoparticles.
The
hydrophobic-hydrophilic nature of zein makes it a good candidate for binding or encapsulating biologically active compounds, such as resveratrol, curcumin, lutein and thymol (Joye, Davidov-Pardo, & McClements, 2015; Wang, Wang, Yang, Guo, & Lin, 2015). We hypothesize that the nanoparticles formed via SMN-Zein interactions can reduce silymarin crystallinity and increase its water solubility. This study evaluated for the first time the development of silymarin-zein (SMN-Zein) nanoparticles/BC nanocomposite films for active food packaging application. The aim of this study were to investigate the effect of the interactions between SMN-Zein nanoparticles and BC nanofibers on chemical and physical properties of the nanocomposite films. SMN-Zein nanoparticles increased the solubility of poorly soluble silymarin and thus improved silymarin release from BC films. Enhancement of the antioxidant and antibacterial activities of SMN-Zein/BC nanocomposite films by active release of silymarin were evaluated. Prevention of lipid oxidation and deterioration in salmon muscle by the active films was determined using total volatile basic nitrogen (TVB-N) and thiobarbituric acid reactive substances (TBARS) measurements.
2. Materials and methods 4
2.1. Materials Trichloroacetic
acid
(TCA),
silymarin,
zein,
malondialdehyde
(MDA),
1,1-diphenyl-2-picrylhydrazyl (DPPH), 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid (ABTS·+) and Folin–Ciocalteu reagent were purchased from Sigma Chemical Co. (USA). 2.2. Preparation of BC films BC sheets were biosynthesized by Gluconacetobacter xylinus (G. xylinus) (BCRC 12335 obtained from FIRDI, Hsinchu, Taiwan) using a static incubation method. Briefly, the bacterial strain was grown on a buffered Schramm and Hestrin (BSH) medium that contained 5 g/l peptone, 1.2 g/l citric acid, 5 g/l yeast, 100 g/l sucrose, and 2.7 g/l Na2HPO4. After incubation at 30 oC for 14 days, a thin layer of BC sheet was formed in the interface of liquid/air. The BC films were washed by deionized water, then boiled with 0.5wt% NaOH for 60 minutes. Finally, the films were washed thoroughly with deionized water until the pH reached 7.0. The BC sheets were lyophilized or dried in air to obtain freeze-dried and air-dried BC films. 2.3. Preparation of SMN-Zein nanoparticles 2.3.1. Preparation of nanoparticles The SMN-Zein nanoparticles were prepared by a liquid antisolvent coprecipitation method. Silymarin and zein were dissolved in 80 v/v % ethanol (5 ml) and heated at 50 oC for 12 h to prepare the stock solutions. The silymarin/zein mixed solutions were subsequently added to the antisolvent (water, 15 ml), under vigorous stirring with a magnetic stirrer at room temperature to obtain the colloidal suspensions of SMN-Zein nanoparticles. The mixtures were heated under reduced pressure using a rotary evaporator to remove ethanol (Buchi R-300, Switzerland). 2.3.2. Characterization of nanoparticles Mean particle diameter and zeta potentials of the SMN-Zein nanoparticles were 5
measured using dynamic light scattering (DLS) on a Zetasizer (Nano ZS, Malvern Instruments, Malvern, UK). Each sample was analyzed in triplet and each replicate was measured eight times to yield the average particle size. SMN-Zein nanoparticle suspensions were dropped onto a 400 mesh copper grid coated with carbon. TEM experiments were performed on a JEM-2200FS microscope (JEOL Ltd, Japan) after drying the samples in air. DPPH and ABTS·+ radicals scavenging activities of SMN-Zein nanoparticles were evaluated using the method as reported in our previous study. (Tang et al., 2013). After a serial dilution of nanoparticle suspensions, 100 µl of the sample were added to 1.9 ml of a 0.1 M DPPH· methanol or 0.75 M ABTS·+ ethanol solutions. Absorbance of the DPPH· and ABTS·+ solutions were measured in triplicate at 517nm and 734 nm to determine half maximal effective concentrations (EC50) for the free radicals scavenging. Hydroxyl radical (OH·) scavenging capacities of SMN-Zein nanoparticles were measured by an ESR spectrometer (EMX-6/1, Bruker, Karlsruhe, Germany). 2.4. Preparation of SMN-Zein/BC nanocomposite films SMN-Zein nanoparticles were prepared according to the above mentioned process. BC films were preswollen in deionized water at 40 oC for 24 h. Then, the films were immersed in the colloidal suspensions of SMN-Zein nanoparticle (200 ml) with shaking (100 rpm) at 30 o
C for 4 h. The obtained SMN-Zein nanoparticle/bacterial cellulose (SMN-Zein/BC)
nanocomposite films were removed from the solution, and repeatedly washed with deionized water. 2.5. Characterization of SMN-Zein/BC nanocomposite films 2.5.1. Scanning electron microscopy (SEM) The film samples were sputter-coated with Au using a sputter coater (IB-2 coater, Hitachi). A Hitachi S-2400 scanning electron microscope was used to detect the morphology of the film samples. All samples were observed with a high-vacuum secondary electron 6
detector at 3 kV (surfaces of film samples) or 7 kV (cross-sections of fractured film samples). 2.5.2. Infrared spectroscopy analysis The dry silymarin, blank zein nanoparticles, and SMN-Zein/BC nanocomposite films were mixed with KBr, respectively. Each mixture was ground into a fine powder and then pressed into a disk. FT-IR spectra were acquired by a Perkin Elmer, Spectrum RXI spectrophotometer. The spectra were scanned in the range of 4000 to 400 cm-1 at a resolution of 4 cm-1. 2.6. Contact angle and water uptake 2.6.1. Contact angle The contact angles of water drops on dried BC films and SMN-Zein/BC nanocomposite films were determined by a contact angle meter (FACE, Model CA-D Type). The contact angle is defined by the angle between the tangent line and the drop baseline. 2.6.2. Water uptake Dried BC film and SMN-Zein/BC nanocomposite films (Wd) were weighed and subsequently immersed into a conical flask containing 250 ml different pH (2.0, 6.0 and 7.4) of swelling medium at room temperature. The samples were collected and the excess swelling medium was removed by blotting with dry paper towels at a predetermined period of time. Afterwards, the films were re-weighed and the swelling ratios at all time points when soaked in the swelling medium were calculated from the following: Swelling ratio = [(Ws/Wd) – 1] Ws and Wd represent the weights of swollen and dry films. 2.7. Drug loading and release SMN-loading contents in the nanocomposite films were shown as total phenol contents (TPC) (Wang et al., 2013). The SMN-Zein/BC nanocomposite films were placed in absolute ethanol and the dissolved SMN was determined by Folin–Ciocalteu reagent. The sample’s 7
absorbance was measured at 765 nm using a Perkin Elmer EnSpire 2300 multimode plate reader (PerkinElmer, Inc., MA, USA) and TPC was calculated by using gallic acid (GA) calibration standard. SMN releases from the nanocomposite films placed in release medium (distilled water) were measured at predetermined intervals by determining the increase of TPC in release medium. 2.8. Dynamic antioxidant capability SMN-Zein/BC nanocomposite films (0.1 g) were placed in 1 liter of distilled water (pH 6.0). At specific time periods, 100 µl of the release medium were adding to 1.9 ml of a 0.1 M DPPH· methanol solution. Dynamic free radical scavenging activities of SMN-Zein/BC nanocomposite films were evaluated by determining the DPPH scavenging ratios according to the previously mentioned methods. 2.9. Antimicrobial testing Staphylococcus aureus (ATCC 6538), Escherichia coli (ATCC 11229) and Pseudomonas aeruginosa (BCRC 10944) purchased from FIRDI (Hsinchu, Taiwan) were grown in nutrient broth. At predetermined time intervals, the bacteria cultures were collected for measuring the optical density (OD595). The antimicrobial test was carried out according to the method previously reported by (Yu et al., 2013). In a 20 ml test tube, a bacteria cultured broth (2×105 cfu/ml, 1 ml) was added and a sterilized film sample (0.1 g) was placed in the broth. After 24 h of incubation, the film sample was removed and then the bacteria cultures were diluted with normal saline and the diluted cultures were grow on nutrient agar plates for 48 h. The formula given below was used to calculate the percentage growth inhibition. Inhibition ratio (%) = (1-CFUsample/CFUcontrol)×100 CFUcontrol or CFUsample represent the colony-forming units of the bacteria culture (CFUcontrol) and the cultures co-incubated in the presence of test films (CFUsample). 2.10. β-carotene bleaching assay 8
An aliquot of 10, 50, 100 μl of SMN-Zein nanoparticles were added into the emulsified system and the mixed was then irradiated with a blue light (410-460 nm) according to the method reported by Yu et al. (2015). At a predetermined time, the absorbance of the samples (As) and control (Ac)
was measured and the inhibition ratio was estimated using the
following equation. Inhibition ratio (%) = [(Asinitial – Asfinal)/(Acinitial – Acfinal)]×100 2.11. Chemical analysis of film-packaged fish Salmon was sliced and 5 grams of the fish muscle was packaged with alkaline-treated BC film and SMN-Zein/BC nanocomposite films and were subsequently stored at 4 oC. The samples were removed from the packages during predetermined periods (day 5, 8, 12 and 16 of storage). Physicochemical analyses were performed as follows at different time intervals to evaluate the quality of the salmon muscle. 2.11.1. Total volatile basic nitrogen TVB-N value was evaluated according to the Conway’s micro diffusion method. Briefly, the fish sample (5 g) was homogenized with 45 ml of TCA 7% (w/v) and TVB-N was measured by steam distillation of the homogenized and filtered fish sample. The volatile base components were collected in 25 ml boric acid (2 %). Finally, the collected samples were titrated with a 0.1 N sulfuric acid solution. The amount of TVB-N was calculated from the consumption of sulfuric acid used for titration. 2.11.2. Thiobarbituric acid reactive substances (TBARS) The fish sample was mixed with aqueous TCA and subsequently homogenized according to the above mentioned procedure. After filtering, the sample (5 ml) was mixed with 5 ml of thiobarbituric acid (20 mM) and incubated in boiling water for 35 min. The sample after cooling was measured at 532 nm by a SPECTRONIC 200, Thermo, USA. The TBARS value was showed as mg malondialdehyde (mg MDA) per kilogram of salmon (kg 9
salmon). 2.12. Statistical analysis All measurements were performed in triplicate and the statistical significance was analyzed by using SAS version 9.1 (SAS Institute, Cary, NC, USA). Data were subjected to analysis of variance (ANOVA), and Duncan’s multiple range tests was carried out for mean comparison. P < 0.05 indicates a statistically significant difference between groups.
3. Results and discussion 3.1. Characterization of BC films Fig. 1A shows the SEM micrographs of native BC films synthesized by G. xylinus and the BC films treated with 0.1N NaOH for 1 h. The size of the fibers in the freeze-dried alkaline-treated BC films was decreased after treatment in an alkaline solution. The decrease in fiber size was attributed to a chain scission reaction occurred in the alkaline solution, which induced breakdown of fiber bundles (McKenna, Mikkelsen, Wehr, Gidley, & Menzies, 2009). The BC film treated with 0.1 N NaOH for 1 h following air-drying shows a highly fibrous but dense packing of ultrafine cellulose nanofibrils. The freeze-dried BC films prepared by the same alkaline treatment showed an interconnected network structure consisting of entangled cellulose nanofibers with diameters less than 100 nm. The space between the adjacent fibers was larger than that of the air-dried BC films. Fig. 1B shows the XRD patterns of native BC film and the alkaline-treated BC films. The freeze-dried, native BC films displayed the typical XRD pattern of cellulose I with three diffraction peaks at 2-theta angles of 14.2°, 16.8° and 22.4° corresponding to the (10), (110), and (200) planes of cellulose (Wu et al., 2012). After treatment with 0.1 N of NaOH solution, the intensity of the peak at 2-theta angle of 16.8° was weaker than that of the native BC film. However, the air-dried BC film retained the peaks at 2-theta angle of 14.2°and 22.4° but the peak at 2-theta 10
angle of 16.8° disappeared. Fig. 1C shows the FT-IR spectrum of native BC films and the alkaline-treated BC films.. The FTIR spectrum of the native BC films displayed characteristic absorption bands at 1560 cm−1 and 1650 cm−1 which were assigned to C=O (amide I) and C-N (amide II) stretch from proteins. After treatment with alkaline, the peak at 1560 cm−1 and 1650 cm−1 decreased obviously, indicating that most of the protein in BC films has been removed. Fig. 1D shows the TGA curves obtained for the film samples. The alkaline-treated BC film shows significant weight loss at around 333.9◦C and 456.5◦C that are associated with the depolymerization of cellulose and the decomposition of glucose units. 3.2. Fabrication and characterization of SMN-Zein nanoparticles Zein is a class of prolamine protein with an isoelectric point at around pH 6.2. Accordingly, the colloidal dispersions containing zein nanoparticles become unstable at pH near the isoelectric point (Joye et al., 2015). The pH-responsive property of the empty zein nanoparticles were characterized by the changes in their hydrodynamic diameter and zeta potential (Fig. 2A). The zeta potential of the empty zein nanoparticles changed from positive to negative by changing pH from 3.0 to 7.0. The hydrodynamic diameters of the empty zein nanoparticles increased obviously at pH 6.0 and 7.0 because of the aggregation of the colloidal dispersion at a pH near the isolectric point (pH 6.2). The empty zein nanoparticles are cationic at pH values lower than 6.0 and anionic at pH 7.0. Because the empty zein nanoparticles and silymarin were stable in weak acid, the silymarin-zein self-assembled nanoparticles were prepared and dispersed in a solution of pH 5.0. The particle sizes of SMN-Zein nanoparticles significantly decreased with the increase of the SMN-to-Zein weight ratio, whereas the zeta potential increase with the increase of the SMN-to-Zein weight ratio (Table 1). SMN0.5-Zein0.5 and SMN1.0-Zein1.0 nanoparticles were selected for further study because they have smaller particle sizes and higher zeta potentials. TEM images show that 11
the SMN0.5-Zein0.5 and SMN1.0-Zein1.0 nanoparticles are spherical with their sizes below 200 nm (Fig. 2B). The interaction of zein and polyphenols caused the structural change that was related to the phenolic groups in zein (Liu, Ma, McClements, & Gao, 2017). The empty zein colloidal nanoparticles showed an absorption at 304 nm that was assigned to its tyrosine residues (Fig. 2C). The SMN-Zein colloidal nanoparticles exhibited considerably lower fluorescence intensities than their empty zein counterpart, suggesting the association of silymarin with the tyrosine groups of zein. Fig. 2D shows the FTIR spectra of SMN, zein and the prepared SMN-Zein nanoparticles. The FTIR spectrum obtained for zein shows the characteristic bands of proteins at 1664 cm-1 and 1545 cm-1, which was assigned to stretching of the carbonyl (C=O, amide I) and the amine (N–H, amide II) of amide bonds. SMN shows the characteristic peaks of flavonolignan ketone vibrations (1639 cm−1), aromatic ring vibrations (in-plane, 821 cm−1), and benzopyran ring vibrations (1084 cm−1). The unique absorption pattern of SMN in the "fingerprint region" falls in the 600 cm-1 to 1400 cm-1 range. However, the FTIR spectrum obtained for SMN-Zein nanoparticles shows the characteristic amide absorption of zein (1693 cm−1 and 1702 cm−1) and the absorptions of SMN in the fingerprint region, indicating that SMN was binding to zein through the formation of hydrogen bonding and hydrophobic interactions (Liu et al., 2017). 3.3. Physical properties of SMN-Zein/BC nanocomposite films The
SMN-Zein/BC
nanocomposite
films
were
prepared
by immersing
the
alkaline-treated BC films in the colloidal suspensions of SMN-Zein nanoparticles. The nanoparticles are firmly attached to BC nanofibers, and then the SMN-Zein/BC composite films were air-dried and freeze-dried (Fig. 3A). From the cross-sectional micrographs of the SMN-Zein/BC nanocomposite films, it is evident that the nanoparticles were firmly adhered to BC nanofibers, leading to the formation of a nanoparticles-coated fiberous structure. As 12
reported by the literatures, the BC nanofiber was negatively charged with a potential value of about −7.5 mV (Lee et al., 2011). SMN0.5-Zein0.5 and SMN1.0-Zein1.0 nanoparticles were both positively charged as indicated by ζ-potential values of 19.7 and 14.9 mV (Table 1). Accordingly, the positively charged SMN-Zein nanoparticles can adhere strongly to the surface of the negatively charged BC nanofibers and deposite on the fibers through their electrostatic interactions. However, SMN nanoparticles alone could not adhere to the BC nanofibers because they were negatively charged (-16.5 mV). Fig. 3B shows the FT-IR spectrum of alkaline-treated BC films and SMN-Zein/BC composite films. The FTIR spectrum of BC nanofiber displayed characteristic absorption bands at 3361 cm−1 and 3235 cm−1 which were assigned to O–H stretch from hydroxyl groups. The peaks at 2914 cm-1 and 2850 cm-1 represented the C–H stretching from
methyl
and
methylene groups. The sugar ring absorptions at 1064 cm-1, 1116 cm-1 and 1236 cm-1 were attributed to the C–O–C stretching vibration of ether and O–H vibrations for the primary and secondary hydroxyl bending. The SMN-Zein/BC nanocomposite films show the sugar ring absorptions from BC nanofiber and the absorptions of SMN in the fingerprint region. The result indicated that SMN-Zein nanoparticles were successfully incorporated in the BC films. The thicknesses of air-dried and freeze-dried BC films were 9.34 ± 2.56 and 50.37 ± 2.77 μm, respectively. Incorporation of SMN0.5-zein0.5 and SMN1.0-zein1.0 nanoparticles into air-dried BC films resulted in the increase of film thickness from 9.34 ± 2.56 μm to 22.79 ± 3.71 and 31.77 ± 0.83 μm. Thickness of freeze-dried BC films incorporated with SMN0.5-Zein0.5 and SMN1.0-Zein1.0 nanoparticles increased from 50.37 ± 2.77 μm to 106.00 ± 1.83 and 154.32 ± 7.93 μm, respectively. The thicker SMN1.0-Zein1.0/BC nanocomposite films represents the incorporation of more nanoparticles in the films, which can be correlated to their higher TPC (117.5 mg GAE/g in SMN1.0-Zein1.0/BC films vs. 69.4 mg GAE/g in SMN0.5-Zein0.5/BC films). Thus, SMN1.0-Zein1.0/BC nanocomposite films were selected for 13
further studies because of their higher TPC contents. Fig. 3C shows the XRD diffraction patterns of BC film and SMN1.0-Zein1.0/BC nanocomposite films. The peaks at 2θ angles of 15.1, 17.2, and 23.2º indicated that BC produced in this work were cellulose I (Vazquez, Foresti, Cerrutti, & Galvagno, 2013). However, the peak intensities of SMN1.0-Zein1.0/BC nanocomposite films at these 2θ angles were similar to that of the pure BC film, indicating that the interaction between SMN-Zein nanoparticles and BC fibers didn’t affect the crystallinity of BC fibers. Wetting properties of BC films and SMN1.0-Zein1.0/BC nanocomposite films were investigated by the contact angle of water droplet on the surface of the films. BC films have highly hydrophilic surfaces, so the water droplet angles of air-dried and freeze-dried BC films were small (28.14º and 24.25º). The contact angle values of the SMN1.0-Zein1.0/BC nanocomposite films increased to 41.50º and 31.62º, indicating that incorporation of SMN-Zein nanoparticles into the BC films increased the hydrophobicity of the film surfaces. Increase of hydrophobicity makes packaging films moisture resistant and provides an excellent barrier against water penetration, which may increase shelf life of food products. 3.4.Swelling and drug release of SMN-Zein/BC nanocomposite films The water uptakes of air-dried and freeze-dried SMN1.0-Zein1.0/BC composite films were measured. As shown in Fig. 4A, the air-dried and freeze-dried BC films reach equilibrium swelling after 1800 minutes. SMN-Zein/BC nanocomposite films demonstrated lower swelling degrees compared to original air-dried and freeze-dried BC films (60.2% and 152.7% vs. 269.8% and 306.9%) because the hydrophobic nature of
the SMN-Zein
nanoparticles that were incorporated in the BC films. There was no significant difference in swelling properties among the BC films incorporated with SMN0.5-Zein0.5 and SMN1.0-Zein1.0 nanoparticles (Fig. 4A). Silymarin is a hydrophobic crystalline compound that has very low solubility in water 14
(0.04 mg/mL). X-ray diffraction study shows that silymarin crystallinity decreased after formation of SMN-Zein nanoparticles (Fig. 4B). An active packaging film is designed for releasing antioxidant and antibacterial compound upon touching the food products in a controlled wet environment. Therefore, distilled water was used as a release medium in the drug release study. Fig. 4C shows that when silymarin powder was directly incorporated into BC films (SMN/BC films), silymarin was released from the films at a slow rate. Only 16.4% of the incprporated silymarin was released form the films after 120 h of release time. The slow release rate was due to the crystalline state and poor aqueous solubility of silymarin. The specific interaction between silymarine and zein results in the formation of well-dispersed SMN-Zein nanoparticles that have a relative larger surface area when compared to silymarin powder (Fig. 3C), thus is beneficial in dissolving silymarin. As shown in Fig. 4C, the air- and freeze-dried SMN-Zein/BC films released silymarin in a much higher ratio than the SMN/BC films, indicating that the nanocomposite films were effective in releasing the active component, silymarin, to improve the antioxidant and antibacterial functions of BC films. The SMN-Zein/BC films placed in the release medium for 24 h showed deformed BC matrix and SMN-Zein nanoparticles due to the release of incorporated silymarin (Fig. 4D). In the initial drug release stage (within 0 to 8 hours), the difference of the silymarin release profiles between SMN/BC films and SMN-Zein/BC films was due to the crystallinity and poor aqueous solubility of silymarin (Fig. 4C). Only a small portion of silymarin molecules that were well dispersed and were not aggregated in the BC films released from the SMN/BC films in the initial stage of drug release. However, after formation of SMN-Zein nanoparticles, the crystallinity of silymarin was decreased. Furthermore, the specific interaction between silymarine and zein results in the formation of well-dispersed SMN-Zein nanoparticles that have a relative larger surface area when compared to silymarin powder, 15
thus is beneficial in dissolving silymarin. The next stage of release showed a slow and sustained release of silymarin from SMN/BC film and SMN-Zein/BC films. Silymarin is a hydrophobic compound and is known to aggregate in water, causing a decreased release rate in the second stage of release. The slow release of silymarin from SMN-Zein/BC films in the second stage of release can be explained by the aggregation of SMN-Zein nanoparticles after immersing the composite films in water, as indicated in the TEM imaging (Fig. 4D). Besides, the freeze-dried SMN-Zein/BC films showed a slightly higher release rate in the second stage of release becaue freeze-drying is known to decrease bulk density of polymeric materials, including BC and zein. As shown in Fig. 4D, after 24 hours immersion in water (with shaking), the SMN-Zein nanoparticles still attached to the BC nanofibers (cross-section). Besides, more than 50% of incorporated silymarin were not released within 120 h of release (Fig. 4C). Silymarin is a flavonoid natural product extracted from the seeds of milk thistle. Silymarin can also be found in some foods and the most common food source is artichokes. Silymarin is generally considered safe and not toxic, and has very poor bioavailability due to its low water solubility. Some studies reported the treatment of various disease with high dosages (＞200 mg/kg/day) of silymarin without obvious side effect (Cetinkunar et al., 2015). In addition, when the films are used for food packaging, they just touch the food products but are not fully immersed in an aqueous solution, which will cause limited release of silymarin. Accordinglly, the composite films would not induce obvious negative effects due to excessive dosages. 3.5. Antioxidant and antibacterial test Previously, we have developed chitosan-based nanoparticles that have antioxidant and antibacterial activities (Yu et al., 2011; Tang et al., 2013). In this study, SMN-Zein nanoparticles were developed for the same purpose. Empty zein nanoparticles showed poor antioxidant property and the EC50 values against DPPH, ABTS·+ and superoxide anion were 16
897.5 ± 21.4 μg/ml, 55.3 ± 2.5 μg/ml and 3213.5 ± 165.7 μg/ml. Silymarin exhibited promising antioxidant potential against a variety of free radicals (Pientaweeratch, Panapisal, & Tansirikongkol, 2016). SMN-Zein nanoparticles with EC50 values of 38.5±1.1 μg/ml against DPPH,·2.4±0.3 μg/ml against ABTS·+, and 214.7±6.9 μg/ml against superoxide anion (Fig. 5A). The signal of the spin adduct (DMPO-OH) decreased by 48.1% under treatment by SMN-Zein nanoparticles (Fig. 5B), suggesting that the nanoparticles have a superior OH· scavenging activity comparable to free SMN (54.5% scavenging ratio). BC films didn't exhibit free radical scavenging properties against DPPH radical (Fig. 5C). However, the air-dried and freeze-dried SMN-Zein/BC nanocomposite films show effective free radicals scavenging capacity. Silymarin incorporated in the air-dried nanocomposite film was effective in scavenging DPPH free radicals for 72 h of antioxidant test. The antioxidant activity of the nanocomposite film could be maintained during long-term storage since antioxidant could be slowly released from the nanocomposite films (Fig. 4B). The free radical scavenging ratio of the freeze-dried nanocomposite film increased rapidly during 6 h and slowly increased thereafter, consequently, its free radical scavenging capacity was lower than that of the air-dried nanocomposite film (Fig. 5C). The antibacterial activity of the alkaline-treated BC films and SMN1.0-Zein1.0/BC nanocomposite films were shown in Fig. 5D. BC films didn’t show any antibacterial activity on the test bacteria (E. coli, S. aureus and P. aeruginosa). Bacterial populations of the nanocomposite films-treated bacterial cultures were significantly less compared to the alkaline-treated BC films-treated groups. The inhibition ratios of the air-dried nanocomposite films against E. coli, S. aureus and P. aeruginosa were 21.6 ± 2.8%, 62.1 ± 1.9%, 28.2 ± 4.9%, respectively. Silymarin has strong activity against Gram-positive bacteria but it is less effective against Gram-negative bacteria (Lee et al., 2003; Evren, & Yurtcu, 2015), therefore the nanocomposite films demonstrate better antibacterial efficacy against S. aureus than 17
against E. coli and P. aeruginosa. The freeze-dried nanocomposite film also shows an inhibitory ratio of 17.4 ± 3.6%, 53.5 ± 2.3% and 23.5 ± 5.4% against E. coli, S. aureus and P. aeruginosa (Fig. 5D), which are slightly less than that of the bacterial culture treated with air-dried nanocomposite film. The results suggested that BC films were endowed with antimicrobial properties after incorporating with SMN-Zein nanoparticles. 3.6. Prevention of spoilage and lipid peroxidation of salmon Carotenoids are known to be bleached under the conditions of deodorization that may involve exposure to oxidant species and blue-violet light irradiation (Yu et al., 2015). This bleaching process involves the cleavage of the double bonds and the formation of carbonyls or epoxides in the final products. SMN1.0-Zein1.0 nanoparticles showed 65% inhibition of oxidation in the β-carotene/linoleic acid/photoirradiation system (Fig. 6A), which is comparable to the inhibitory efficiency of RES1.0-Zein1.0 nanoparticles. Resveratrol (RES) is an effective antioxidant and the preparation of RES-Zein nanoparticles has been well studied (Joye et al., 2015). The results suggest that SMN1.0-Zein1.0 nanoparticles have an effective antioxidant activity that can protect β-carotene against linoleic acid peroxidation and photoirradiation-induced deodorization. Next, SMN1.0-Zein1.0 nanoparticles were incorporated into BC films and the effect of SMN1.0-Zein1.0/BC films packaging on the changes in TVNB values of salmon fillets during refrigerated storage were examined (Fig. 6B). The TVB-N values of the refrigerated salmon fillet in control group continuously increased (p < 0.05) from 7.65 mg N/100 g salmon to 66.6 mg N/100 g salmon fillet as the storage time increased to 16 days. When salmon fillets were packaged with SMN1.0-Zein1.0/BC nanocomposite films, the TVB-N contents were kept at lower levels for five days, compared with the control and the salmon fillets packaged with BC films. The TVB-N content of salmon packaged with air-dried SMN1.0-zein1.0/BC nanocomposite film was still below the TVB-N limit (30 mg N/100 g fish) after 12 days of 18
storage (Fig. 6B). Antibacterial and antiadherent/antibiofilm activity of silymarin against certain bacteria and fungi could reduce the production of volatile amines caused by microbial degradation of proteins (de Oliveira et al., 2015; Evren, & Yurtcu, 2015). The TBARS value of uncoated salmon muscle increased from 0.92 to 4.42 mg MDA/kg sample during after storage of 16 days due to lipid oxidation (Fig. 6C). The freeze-dried BC films didn’t significantly reduced lipid oxidation in salmon muscle compared to the uncoated salmon
(control
group).
However,
air-dried
and
freeze-dried
SMN1.0-Zein1.0/BC
nanocomposite films inhibited lipid peroxidation and the effects were significant (2.43 mg and 3.38 mg MDA/kg sample) compared to control (4.42 mg MDA/kg sample). Our previous study showed that caffeic acid-incorporated active films effectively inhibited lipid oxidation in fish oil emulsions (Yu et al., 2013). The protective effect of the SMN-Zein/BC films is attributed to the reason that silymarin can release from the films and exert a promising antioxidant potential against lipid oxidation (Pientaweeratch et al., 2016; Razavi-Azarkhiavi et al., 2014). Conclusion SMN-Zein/BC nanocomposite films were prepared by incorporation of SMN-Zein nanoparticles in BC nanofiber matrix. Antimicrobial and antioxidant activity of
BC films
were enhanced with the incorporation of bioactive SMN-Zein nanoparticles. The results demonstrated the efficacy the air-dried SMN-Zein/BC nanocomposite film in preventing the deterioration of salmon muscle and slowing down the lipid oxidation, thus allowing the packaged fish with extended shelf life in cold storage. Acknowledgments The authors gratefully acknowledge the financial support provided by the National Science Council, Taiwan, Republic of China (NSC 101-2221-E-038-016-MY3 and MOST 103-2313-B-019-003-MY3). 19
Reference Abeer, M. M., Amin, M. C. I. M., & Martin, C. (2014). A review of bacterial cellulose-based drug delivery systems: their biochemistry, current approaches and future prospects. Journal of Pharmacy and Pharmacology, 66, 1047-1061. Cetinkunar, S., Tokgoz, S., Bilgin, B. C., Erdem, H., Aktimur, R., Can, S., Erol, H. S., Isgoren, A., Sozen, S., & Polat, Y. (2015). The effect of silymarin on hepatic regeneration after partial hepatectomy: is silymarin effective in hepatic regeneration? International Journal of Clinical and Experimental Medicine, 8, 2578-2585. de Oliveira, D. R., Tintino, S. R., Braga, M. F. B. M., Boligon, A. A., Athayde, M. L., Coutinho, H. D. M., de Menezes, I. R. A., & Fachinetto, R. (2015). In vitro antimicrobial and modulatory activity of the natural products silymarin and silibinin. BioMed Research International, 2015, 1-7. Evren, E., & Yurtcu, E. (2015). In vitro effects on biofilm viability and antibacterial and antiadherent activities of silymarin. Folia Microbiologica, 60, 351-356. Gao, A. Q., Zhang, C., Song, K. L., & Hou, A. Q. (2014). Preparation of multi-functional cellulose containing huge conjugated system and its UV-protective and antibacterial property. Carbohydrate Polymers, 114, 392-398. Janpetch, N., Saito, N., & Rujiravanit, R. (2016). Fabrication of bacterial cellulose-ZnO composite via solution plasma process for antibacterial applications. Carbohydrate 20
Polymers, 148, 335-344. Jebel, F. S., & Almasi, H. (2016). Morphological, physical, antimicrobial and release properties of ZnO nanoparticles-loaded bacterial cellulose films. Carbohydrate Polymers, 149, 8-19. Joye, I. J., Davidov-Pardo, G., & McClements, D. J. (2015). Encapsulation of resveratrol in biopolymer particles produced using liquid antisolvent precipitation. Part 2: Stability and functionality. Food Hydrocolloids, 49, 127-134. Lee, D. G., Kim, H. K., Park, Y., Park, S. C., Woo, E. R., Jeong, H. G., & Hahm, K. S. (2003). Gram-positive bacteria specific properties of silybin derived from Silybum marianum. Archives of Pharmacal Research, 26, 597-600. Lee, K. Y., Quero, F., Blaker, J. J., Hill, C. A. S., Eichhorn, S. J., & Bismarck, A. (2011). Surface only modification of bacterial cellulose nanofibres with organic acids. Cellulose, 18, 595-605. Liu, F. G., Ma, C. C., McClements, D. J., & Gao, Y. X. (2017). A comparative study of covalent and non-covalent interactions between zein and polyphenols in ethanol-water solution. Food Hydrocolloids, 63, 625-634. McKenna, B. A., Mikkelsen, D., Wehr, J. B., Gidley, M. J., & Menzies, N. W. (2009). Mechanical and structural properties of native and alkali-treated bacterial cellulose produced by Gluconacetobacter xylinus strain ATCC 53524. Cellulose, 16, 21
1047-1055. Pientaweeratch, S., Panapisal, V., & Tansirikongkol, A. (2016). Antioxidant, anti-collagenase and anti-elastase activities of Phyllanthus emblica, Manilkara zapota and silymarin: an in vitro comparative study for anti-aging applications. Pharmaceutical Biology, 54, 1865-1872. Razavi-Azarkhiavi, K., Ali-Omrani, M., Solgi, R., Bagheri, P., Haji-Noormohammadi, M., Amani, N., & Sepand, M. R. (2014). Silymarin alleviates bleomycin-induced pulmonary toxicity and lipid peroxidation in mice. Pharmaceutical Biology, 52, 1267-1271. Shao, W., Liu, H., Wang, S. X., Wu, J. M., Huang, M., Min, H. H., & Liu, X. F. (2016). Controlled release and antibacterial activity of tetracycline hydrochloride-loaded bacterial cellulose composite membranes. Carbohydrate Polymers, 145, 114-120 Sukhtezari, S., Almasi, H., Pirsa, S., Zandi, M., & Pirouzifard, M. (2017). Development of bacterial cellulose based slow-release active films by incorporation of Scrophularia striata Boiss. extract. Carbohydrate Polymers, 156, 340-350. Tang, D. W., Yu, S. H., Ho, Y. C., Huang, B. Q., Tsai, G. J., Hsieh, H. Y., Sung, H. W., & Mi, F. L. (2013). Characterization of tea catechins-loaded nanoparticles prepared from chitosan and an edible polypeptide. Food Hydrocolloids, 30, 33-41. Theodosiou, E., Purchartova, K., Stamatis, H., Kolisis, F., & Kren, V. (2014). Bioavailability 22
of silymarin flavonolignans: drug formulations and biotransformation. Phytochemistry Reviews, 13, 1-18. Vazquez, A., Foresti, M. L., Cerrutti, P., & Galvagno, M. (2013). Bacterial Cellulose from Simple and Low Cost Production Media by Gluconacetobacter xylinus. Journal of Polymers and the Environment, 21, 545-554. Wang, Y. H., Wang, J. M., Yang, X. Q., Guo, J., & Lin, Y. (2015). Amphiphilic zein hydrolysate as a novel nano-delivery vehicle for curcumin. Food & Function, 6, 2636-2645. Wu, J., Zheng, Y. D., Yang, Z., Cui, Q. Y., Wang, Q. L., Gao, S., & Ding, X. (2012). Chemical modifications and characteristic changes in bacterial cellulose treated with different media. Journal of Polymer Research, 19, 9945. Yu, S. H., Mi, F. L., Pang, J. C., Jiang, S. C., Kuo, T. H., Wu, S. J., & Shyu, S. S. (2011). Preparation and characterization of radical and pH-responsive chitosan-gallic acid conjugate drug carriers. Carbohydrate Polymers, 84, 794-802. Yu, S. H., Hsieh, H. Y., Pang, J. C., Tang, D. W., Shih, C. M., Tsai, M. L., Tsai, Y. C., & Mi, F. L. (2013). Active films from water soluble chitosan/cellulose composites incorporating releasable caffeic acid for inhibition of lipid oxidation in fish oil emulsions. Food Hydrocolloids, 32, 9-19. Yu, S. H., Tsai, M. L., Lin, B. X., Lin, C. W., & Mi, F. L. (2015). Tea catechins-cross-linked 23
methylcellulose active films for inhibition of light irradiation and lipid peroxidation induced beta-carotene degradation. Food Hydrocolloids, 44, 491-505. Yun, D. G., & Lee, D. G. (2017). Silymarin exerts antifungal effects via membrane-targeted mode of action by increasing permeability and inducing oxidative stress. Biochimica et Biophysica Acta, 1859, 467-474.
Figure captions Fig. 1. Characterization of native and alkaline-treated BC films: (A) SEM micrograph, (B) XRD pattern, (C) FTIR spectra, (D) TGA and DTG curves . Fig. 2. (A) Influence of pH on the mean particle size and zeta potential of SMN1.0-Zein1.0 nanoparticles, (B) TEM micrographs of SMN1.0-Zein1.0 (a) and SMN0.5-Zein0.5 (b) nanoparticles, (C) fluorescence spectra of zein and SMN-Zein nanoparticles, (D) FTIR spectra of zein, SMN and the prepared SMN-Zein nanoparticles. Fig. 3. Characterization of SMN-Zein/BC nanocomposite films: (A) SEM micrograph, (B) 24
FT-IR spectra, (C) XRD pattern, (D) water drop contact angle. Fig. 4. (A) Swelling properties of BC films and SMN1.0-Zein1.0/BC nanocomposite films, (B) XRD pattern of SMN powder and crystal, and SMN-Zein nanoparticles, (C) silymarin release profiles from SMN1.0-Zein1.0/BC nanocomposite films, (D) SEM micrographs of SMN1.0-Zein1.0/BC nanocomposite films after 24 h placed in release medium. Fig. 5. Antioxidant and antibacterial properties of SMN1.0-Zein1.0/BC nanocomposite films: (A) EC50 of DPPH free radical scavenging activity, (B) EPR spectra of DMPO spin adducts of hydroxyl radical, (C) the continuous DPPH free radical scavenging behavior, (D) inhibition ratio of the nanocomposite films against E. coli, S. aureus and P. aeruginosa Fig. 6 Antioxidant and antibacterial properties of SMN1.0-Zein1.0/BC nanocomposite films: (A) EC50 of DPPH free radical scavenging activity, (B) EPR spectra of DMPO spin adducts of hydroxyl radical, (C) the continuous DPPH free radical scavenging behavior, (D) inhibition ratio of the nanocomposite films against E. coli, S. aureus and P. aeruginosa
25
26
27
28
29
30
31
Table 1. Mean particle sizes, zeta potentials and SMN loading efficiency of SMN-Zein nanoparticles. silymarin:zein
mean particle size
zeta potential
loading efficiency
(wt %/wt %)
(nm)
(mV)
(%)
0.50:0.20
2898.5 ± 38.3
–
–
0.50:0.30
767.5 ± 15.6
25.4 ± 0.3
61.5 ± 4.2
0.50:0.40
228.7 ± 6.8
23.9 ± 0.6
68.8 ± 5.1
Siymarin 0.5 wt%
32
0.50:0.50
198.5 ± 9.8
19.7 ± 0.8
73.4 ± 3.8
1.00:0.70
1690.5 ± 45.2
–
–
1.00:0.80
526.7 ± 3.8
20.5 ± 0.7
85.5 ± 5.7
1.00:0.90
229.8 ± 4.7
17.7 ± 0.5
87.2 ± 3.9
1.00:1.00
214.4 ± 1.4
14.9 ± 0.6
94.3 ± 2.3
Siymarin 1.0 wt%
33
S0144-8617(17)31142-6 https://doi.org/10.1016/j.carbpol.2017.09.100 CARP 12845
To appear in: Received date: Revised date: Accepted date:
8-8-2017 28-9-2017 29-9-2017
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Drug release and antioxidant/antibacterial activities of silymarin-zein nanoparticle/bacterial cellulose nanofiber composite films
Yi-Hsuan Tsai1, Yu-Ning Yang1, Yi-Cheng Ho2, Min-Lang Tsai1*, Fwu-Long Mi3,4,5*
1. Department of Food Science, National Taiwan Ocean University, Keelung 20224, Taiwan, ROC 2. Department of Bioagriculture Science, National Chiayi University, Chiayi 60004, Taiwan. 3. Department of Biochemistry and Molecular Cell Biology, School of Medicine, College of Medicine, Taipei Medical University, Taipei 11031, Taiwan, ROC 4. Graduate Institute of Medical Sciences, College of Medicine, Taipei Medical University, Taipei 110, Taiwan 5. Graduate Institute of Nanomedicine and Medical Engineering, College of Biomedical Engineering, Taipei Medical University, Taipei 11031, Taiwan
Correspondence to: Fwu-Long Mi, PhD Professor Department of Biochemistry School of Medicine College of Medicine Taipei Medical University Taipei, Taiwan 110, ROC Fax: 886-2-2735-6689 E-mail: [email protected] The two corresponding authors (Min-Lang Tsai1* and Fwu-Long Mi2,5*) contributed equally to this work
1
ABSTRACT Bacterial cellulose (BC) is a biopolymer composed of nanofibers which has excellent film-forming ability. However, BC do not have antibacterial or antioxidant activity, thus limiting the applicability of BC for food and biomedical applications. In this study, flavonoid silymarin (SMN) and zein were assembled into spherical SMN-Zein nanoparticles that could be effectively adsorbed onto BC nanofibers. SMN-Zein nanoparticles greatly changed the wettability
and
swelling
property
of
BC
films
due
to
the
formation
of
nanoparticles/nanofibers nanocomposites. SMN-Zein nanoparticles ehanced the release of sparingly soluble silymarin from the nanocomposite films. The active films showed more effective antioxidant and antibacterial activities as compared with pure BC films and thus were able to protect salmon muscle from deterioration and lipid oxidation. These findings suggest that the nanoparticle/nanofiber composites may offer a suitable platform for modification of BC films with improved drug release properties and biological activities. Keywords: bacterial cellulose, zein, drug release, active films, antibacterial, antioxidant
2
1. Introduction Bacterial cellulose (BC) is a biopolymer that can be synthesized by Acetobacter xylinum, which receives growing interest due to its higher biocompatibility, water holding capacity and porosity. BC has become a promising material for biomedical applications, including drug delivery, wound healing and tissue regeneration (Abeer, Amin, & Martin, 2014). BC has excellent film-forming and emulsion-stabilizing properties, making it suitable for use in food applications, such as gelling agents, food stabilizer and food packaging materials. Lipid oxidation and microbiological spoilage are the major causes of quality deterioration in food products. Unfortunately, BC films do not have any antibacterial or antioxidant activity, thus it should be incorporated with active ingredients for food preservation or biomedical purposes. BC films incorporating antioxidants or antimicrobials such as, benzoic acid derivatives, tetracycline and ε-polylysine have been developed to overcome the obstacles (Gao, Zhang, Song, & Hou, 2014; Shao et al., 2016; Sukhtezari, Almasi, Pirsa, Zandi, & Pirouzifard, 2017). Flavonoids is one of the most abundant naturally occurring products can inhibit the growth of microorganisms and protect cells from oxidative stress. Silymarin, a naturally occurring flavonoid isolated from Silybum marianum (L.) Gaertn. (milk thistle), has antioxidant activity against lipid peroxidation (Razavi-Azarkhiavi et al., 2014). Silymarin also demonstrated antimicrobial activity against pathogenic fungi, viruses and bacteria (Yun, & Lee, 2017; Lee, et al., 2003) and exhibited antiadherent/antibiofilm activity against S. epidermidis (Evren, & Yurtcu, 2015). However, silymarin is poorly soluble in water, causing the low availability of silymarin. Various methods have been developed with the aim to enhance aqueous solubility and bioavailability of silymarin, such as microemulsion, proliposome, poly(ethylene glycol) 400 (PEG 400) and phospholipid-based complexes (Theodosiou, Purchartova, Stamatis, Kolisis, & Kren, 2014). 3
In the development of a BC-based composite film, scaffold, wound dressing or food packaging, it is a challenge to incorporate a water-insoluble active compound into the BC matrix and then controlled the release of this active ingredient. Antibacterial BC composites were developed by the incorporation of silver or zinc oxide nanoparticles (Janpetch, Saito, & Rujiravanit, 2016; Jebel, & Almasi, 2016), yet these BC-based nanocomposites can’t release active compounds to exert antioxidant and anti-inflammatory effects. To overcome the problems, we developed a new type of antioxidant and antibacterial BC nanocomposites by incorporating
BC
films
with
silymarin-zein
(SMN-Zein)
nanoparticles.
The
hydrophobic-hydrophilic nature of zein makes it a good candidate for binding or encapsulating biologically active compounds, such as resveratrol, curcumin, lutein and thymol (Joye, Davidov-Pardo, & McClements, 2015; Wang, Wang, Yang, Guo, & Lin, 2015). We hypothesize that the nanoparticles formed via SMN-Zein interactions can reduce silymarin crystallinity and increase its water solubility. This study evaluated for the first time the development of silymarin-zein (SMN-Zein) nanoparticles/BC nanocomposite films for active food packaging application. The aim of this study were to investigate the effect of the interactions between SMN-Zein nanoparticles and BC nanofibers on chemical and physical properties of the nanocomposite films. SMN-Zein nanoparticles increased the solubility of poorly soluble silymarin and thus improved silymarin release from BC films. Enhancement of the antioxidant and antibacterial activities of SMN-Zein/BC nanocomposite films by active release of silymarin were evaluated. Prevention of lipid oxidation and deterioration in salmon muscle by the active films was determined using total volatile basic nitrogen (TVB-N) and thiobarbituric acid reactive substances (TBARS) measurements.
2. Materials and methods 4
2.1. Materials Trichloroacetic
acid
(TCA),
silymarin,
zein,
malondialdehyde
(MDA),
1,1-diphenyl-2-picrylhydrazyl (DPPH), 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid (ABTS·+) and Folin–Ciocalteu reagent were purchased from Sigma Chemical Co. (USA). 2.2. Preparation of BC films BC sheets were biosynthesized by Gluconacetobacter xylinus (G. xylinus) (BCRC 12335 obtained from FIRDI, Hsinchu, Taiwan) using a static incubation method. Briefly, the bacterial strain was grown on a buffered Schramm and Hestrin (BSH) medium that contained 5 g/l peptone, 1.2 g/l citric acid, 5 g/l yeast, 100 g/l sucrose, and 2.7 g/l Na2HPO4. After incubation at 30 oC for 14 days, a thin layer of BC sheet was formed in the interface of liquid/air. The BC films were washed by deionized water, then boiled with 0.5wt% NaOH for 60 minutes. Finally, the films were washed thoroughly with deionized water until the pH reached 7.0. The BC sheets were lyophilized or dried in air to obtain freeze-dried and air-dried BC films. 2.3. Preparation of SMN-Zein nanoparticles 2.3.1. Preparation of nanoparticles The SMN-Zein nanoparticles were prepared by a liquid antisolvent coprecipitation method. Silymarin and zein were dissolved in 80 v/v % ethanol (5 ml) and heated at 50 oC for 12 h to prepare the stock solutions. The silymarin/zein mixed solutions were subsequently added to the antisolvent (water, 15 ml), under vigorous stirring with a magnetic stirrer at room temperature to obtain the colloidal suspensions of SMN-Zein nanoparticles. The mixtures were heated under reduced pressure using a rotary evaporator to remove ethanol (Buchi R-300, Switzerland). 2.3.2. Characterization of nanoparticles Mean particle diameter and zeta potentials of the SMN-Zein nanoparticles were 5
measured using dynamic light scattering (DLS) on a Zetasizer (Nano ZS, Malvern Instruments, Malvern, UK). Each sample was analyzed in triplet and each replicate was measured eight times to yield the average particle size. SMN-Zein nanoparticle suspensions were dropped onto a 400 mesh copper grid coated with carbon. TEM experiments were performed on a JEM-2200FS microscope (JEOL Ltd, Japan) after drying the samples in air. DPPH and ABTS·+ radicals scavenging activities of SMN-Zein nanoparticles were evaluated using the method as reported in our previous study. (Tang et al., 2013). After a serial dilution of nanoparticle suspensions, 100 µl of the sample were added to 1.9 ml of a 0.1 M DPPH· methanol or 0.75 M ABTS·+ ethanol solutions. Absorbance of the DPPH· and ABTS·+ solutions were measured in triplicate at 517nm and 734 nm to determine half maximal effective concentrations (EC50) for the free radicals scavenging. Hydroxyl radical (OH·) scavenging capacities of SMN-Zein nanoparticles were measured by an ESR spectrometer (EMX-6/1, Bruker, Karlsruhe, Germany). 2.4. Preparation of SMN-Zein/BC nanocomposite films SMN-Zein nanoparticles were prepared according to the above mentioned process. BC films were preswollen in deionized water at 40 oC for 24 h. Then, the films were immersed in the colloidal suspensions of SMN-Zein nanoparticle (200 ml) with shaking (100 rpm) at 30 o
C for 4 h. The obtained SMN-Zein nanoparticle/bacterial cellulose (SMN-Zein/BC)
nanocomposite films were removed from the solution, and repeatedly washed with deionized water. 2.5. Characterization of SMN-Zein/BC nanocomposite films 2.5.1. Scanning electron microscopy (SEM) The film samples were sputter-coated with Au using a sputter coater (IB-2 coater, Hitachi). A Hitachi S-2400 scanning electron microscope was used to detect the morphology of the film samples. All samples were observed with a high-vacuum secondary electron 6
detector at 3 kV (surfaces of film samples) or 7 kV (cross-sections of fractured film samples). 2.5.2. Infrared spectroscopy analysis The dry silymarin, blank zein nanoparticles, and SMN-Zein/BC nanocomposite films were mixed with KBr, respectively. Each mixture was ground into a fine powder and then pressed into a disk. FT-IR spectra were acquired by a Perkin Elmer, Spectrum RXI spectrophotometer. The spectra were scanned in the range of 4000 to 400 cm-1 at a resolution of 4 cm-1. 2.6. Contact angle and water uptake 2.6.1. Contact angle The contact angles of water drops on dried BC films and SMN-Zein/BC nanocomposite films were determined by a contact angle meter (FACE, Model CA-D Type). The contact angle is defined by the angle between the tangent line and the drop baseline. 2.6.2. Water uptake Dried BC film and SMN-Zein/BC nanocomposite films (Wd) were weighed and subsequently immersed into a conical flask containing 250 ml different pH (2.0, 6.0 and 7.4) of swelling medium at room temperature. The samples were collected and the excess swelling medium was removed by blotting with dry paper towels at a predetermined period of time. Afterwards, the films were re-weighed and the swelling ratios at all time points when soaked in the swelling medium were calculated from the following: Swelling ratio = [(Ws/Wd) – 1] Ws and Wd represent the weights of swollen and dry films. 2.7. Drug loading and release SMN-loading contents in the nanocomposite films were shown as total phenol contents (TPC) (Wang et al., 2013). The SMN-Zein/BC nanocomposite films were placed in absolute ethanol and the dissolved SMN was determined by Folin–Ciocalteu reagent. The sample’s 7
absorbance was measured at 765 nm using a Perkin Elmer EnSpire 2300 multimode plate reader (PerkinElmer, Inc., MA, USA) and TPC was calculated by using gallic acid (GA) calibration standard. SMN releases from the nanocomposite films placed in release medium (distilled water) were measured at predetermined intervals by determining the increase of TPC in release medium. 2.8. Dynamic antioxidant capability SMN-Zein/BC nanocomposite films (0.1 g) were placed in 1 liter of distilled water (pH 6.0). At specific time periods, 100 µl of the release medium were adding to 1.9 ml of a 0.1 M DPPH· methanol solution. Dynamic free radical scavenging activities of SMN-Zein/BC nanocomposite films were evaluated by determining the DPPH scavenging ratios according to the previously mentioned methods. 2.9. Antimicrobial testing Staphylococcus aureus (ATCC 6538), Escherichia coli (ATCC 11229) and Pseudomonas aeruginosa (BCRC 10944) purchased from FIRDI (Hsinchu, Taiwan) were grown in nutrient broth. At predetermined time intervals, the bacteria cultures were collected for measuring the optical density (OD595). The antimicrobial test was carried out according to the method previously reported by (Yu et al., 2013). In a 20 ml test tube, a bacteria cultured broth (2×105 cfu/ml, 1 ml) was added and a sterilized film sample (0.1 g) was placed in the broth. After 24 h of incubation, the film sample was removed and then the bacteria cultures were diluted with normal saline and the diluted cultures were grow on nutrient agar plates for 48 h. The formula given below was used to calculate the percentage growth inhibition. Inhibition ratio (%) = (1-CFUsample/CFUcontrol)×100 CFUcontrol or CFUsample represent the colony-forming units of the bacteria culture (CFUcontrol) and the cultures co-incubated in the presence of test films (CFUsample). 2.10. β-carotene bleaching assay 8
An aliquot of 10, 50, 100 μl of SMN-Zein nanoparticles were added into the emulsified system and the mixed was then irradiated with a blue light (410-460 nm) according to the method reported by Yu et al. (2015). At a predetermined time, the absorbance of the samples (As) and control (Ac)
was measured and the inhibition ratio was estimated using the
following equation. Inhibition ratio (%) = [(Asinitial – Asfinal)/(Acinitial – Acfinal)]×100 2.11. Chemical analysis of film-packaged fish Salmon was sliced and 5 grams of the fish muscle was packaged with alkaline-treated BC film and SMN-Zein/BC nanocomposite films and were subsequently stored at 4 oC. The samples were removed from the packages during predetermined periods (day 5, 8, 12 and 16 of storage). Physicochemical analyses were performed as follows at different time intervals to evaluate the quality of the salmon muscle. 2.11.1. Total volatile basic nitrogen TVB-N value was evaluated according to the Conway’s micro diffusion method. Briefly, the fish sample (5 g) was homogenized with 45 ml of TCA 7% (w/v) and TVB-N was measured by steam distillation of the homogenized and filtered fish sample. The volatile base components were collected in 25 ml boric acid (2 %). Finally, the collected samples were titrated with a 0.1 N sulfuric acid solution. The amount of TVB-N was calculated from the consumption of sulfuric acid used for titration. 2.11.2. Thiobarbituric acid reactive substances (TBARS) The fish sample was mixed with aqueous TCA and subsequently homogenized according to the above mentioned procedure. After filtering, the sample (5 ml) was mixed with 5 ml of thiobarbituric acid (20 mM) and incubated in boiling water for 35 min. The sample after cooling was measured at 532 nm by a SPECTRONIC 200, Thermo, USA. The TBARS value was showed as mg malondialdehyde (mg MDA) per kilogram of salmon (kg 9
salmon). 2.12. Statistical analysis All measurements were performed in triplicate and the statistical significance was analyzed by using SAS version 9.1 (SAS Institute, Cary, NC, USA). Data were subjected to analysis of variance (ANOVA), and Duncan’s multiple range tests was carried out for mean comparison. P < 0.05 indicates a statistically significant difference between groups.
3. Results and discussion 3.1. Characterization of BC films Fig. 1A shows the SEM micrographs of native BC films synthesized by G. xylinus and the BC films treated with 0.1N NaOH for 1 h. The size of the fibers in the freeze-dried alkaline-treated BC films was decreased after treatment in an alkaline solution. The decrease in fiber size was attributed to a chain scission reaction occurred in the alkaline solution, which induced breakdown of fiber bundles (McKenna, Mikkelsen, Wehr, Gidley, & Menzies, 2009). The BC film treated with 0.1 N NaOH for 1 h following air-drying shows a highly fibrous but dense packing of ultrafine cellulose nanofibrils. The freeze-dried BC films prepared by the same alkaline treatment showed an interconnected network structure consisting of entangled cellulose nanofibers with diameters less than 100 nm. The space between the adjacent fibers was larger than that of the air-dried BC films. Fig. 1B shows the XRD patterns of native BC film and the alkaline-treated BC films. The freeze-dried, native BC films displayed the typical XRD pattern of cellulose I with three diffraction peaks at 2-theta angles of 14.2°, 16.8° and 22.4° corresponding to the (10), (110), and (200) planes of cellulose (Wu et al., 2012). After treatment with 0.1 N of NaOH solution, the intensity of the peak at 2-theta angle of 16.8° was weaker than that of the native BC film. However, the air-dried BC film retained the peaks at 2-theta angle of 14.2°and 22.4° but the peak at 2-theta 10
angle of 16.8° disappeared. Fig. 1C shows the FT-IR spectrum of native BC films and the alkaline-treated BC films.. The FTIR spectrum of the native BC films displayed characteristic absorption bands at 1560 cm−1 and 1650 cm−1 which were assigned to C=O (amide I) and C-N (amide II) stretch from proteins. After treatment with alkaline, the peak at 1560 cm−1 and 1650 cm−1 decreased obviously, indicating that most of the protein in BC films has been removed. Fig. 1D shows the TGA curves obtained for the film samples. The alkaline-treated BC film shows significant weight loss at around 333.9◦C and 456.5◦C that are associated with the depolymerization of cellulose and the decomposition of glucose units. 3.2. Fabrication and characterization of SMN-Zein nanoparticles Zein is a class of prolamine protein with an isoelectric point at around pH 6.2. Accordingly, the colloidal dispersions containing zein nanoparticles become unstable at pH near the isoelectric point (Joye et al., 2015). The pH-responsive property of the empty zein nanoparticles were characterized by the changes in their hydrodynamic diameter and zeta potential (Fig. 2A). The zeta potential of the empty zein nanoparticles changed from positive to negative by changing pH from 3.0 to 7.0. The hydrodynamic diameters of the empty zein nanoparticles increased obviously at pH 6.0 and 7.0 because of the aggregation of the colloidal dispersion at a pH near the isolectric point (pH 6.2). The empty zein nanoparticles are cationic at pH values lower than 6.0 and anionic at pH 7.0. Because the empty zein nanoparticles and silymarin were stable in weak acid, the silymarin-zein self-assembled nanoparticles were prepared and dispersed in a solution of pH 5.0. The particle sizes of SMN-Zein nanoparticles significantly decreased with the increase of the SMN-to-Zein weight ratio, whereas the zeta potential increase with the increase of the SMN-to-Zein weight ratio (Table 1). SMN0.5-Zein0.5 and SMN1.0-Zein1.0 nanoparticles were selected for further study because they have smaller particle sizes and higher zeta potentials. TEM images show that 11
the SMN0.5-Zein0.5 and SMN1.0-Zein1.0 nanoparticles are spherical with their sizes below 200 nm (Fig. 2B). The interaction of zein and polyphenols caused the structural change that was related to the phenolic groups in zein (Liu, Ma, McClements, & Gao, 2017). The empty zein colloidal nanoparticles showed an absorption at 304 nm that was assigned to its tyrosine residues (Fig. 2C). The SMN-Zein colloidal nanoparticles exhibited considerably lower fluorescence intensities than their empty zein counterpart, suggesting the association of silymarin with the tyrosine groups of zein. Fig. 2D shows the FTIR spectra of SMN, zein and the prepared SMN-Zein nanoparticles. The FTIR spectrum obtained for zein shows the characteristic bands of proteins at 1664 cm-1 and 1545 cm-1, which was assigned to stretching of the carbonyl (C=O, amide I) and the amine (N–H, amide II) of amide bonds. SMN shows the characteristic peaks of flavonolignan ketone vibrations (1639 cm−1), aromatic ring vibrations (in-plane, 821 cm−1), and benzopyran ring vibrations (1084 cm−1). The unique absorption pattern of SMN in the "fingerprint region" falls in the 600 cm-1 to 1400 cm-1 range. However, the FTIR spectrum obtained for SMN-Zein nanoparticles shows the characteristic amide absorption of zein (1693 cm−1 and 1702 cm−1) and the absorptions of SMN in the fingerprint region, indicating that SMN was binding to zein through the formation of hydrogen bonding and hydrophobic interactions (Liu et al., 2017). 3.3. Physical properties of SMN-Zein/BC nanocomposite films The
SMN-Zein/BC
nanocomposite
films
were
prepared
by immersing
the
alkaline-treated BC films in the colloidal suspensions of SMN-Zein nanoparticles. The nanoparticles are firmly attached to BC nanofibers, and then the SMN-Zein/BC composite films were air-dried and freeze-dried (Fig. 3A). From the cross-sectional micrographs of the SMN-Zein/BC nanocomposite films, it is evident that the nanoparticles were firmly adhered to BC nanofibers, leading to the formation of a nanoparticles-coated fiberous structure. As 12
reported by the literatures, the BC nanofiber was negatively charged with a potential value of about −7.5 mV (Lee et al., 2011). SMN0.5-Zein0.5 and SMN1.0-Zein1.0 nanoparticles were both positively charged as indicated by ζ-potential values of 19.7 and 14.9 mV (Table 1). Accordingly, the positively charged SMN-Zein nanoparticles can adhere strongly to the surface of the negatively charged BC nanofibers and deposite on the fibers through their electrostatic interactions. However, SMN nanoparticles alone could not adhere to the BC nanofibers because they were negatively charged (-16.5 mV). Fig. 3B shows the FT-IR spectrum of alkaline-treated BC films and SMN-Zein/BC composite films. The FTIR spectrum of BC nanofiber displayed characteristic absorption bands at 3361 cm−1 and 3235 cm−1 which were assigned to O–H stretch from hydroxyl groups. The peaks at 2914 cm-1 and 2850 cm-1 represented the C–H stretching from
methyl
and
methylene groups. The sugar ring absorptions at 1064 cm-1, 1116 cm-1 and 1236 cm-1 were attributed to the C–O–C stretching vibration of ether and O–H vibrations for the primary and secondary hydroxyl bending. The SMN-Zein/BC nanocomposite films show the sugar ring absorptions from BC nanofiber and the absorptions of SMN in the fingerprint region. The result indicated that SMN-Zein nanoparticles were successfully incorporated in the BC films. The thicknesses of air-dried and freeze-dried BC films were 9.34 ± 2.56 and 50.37 ± 2.77 μm, respectively. Incorporation of SMN0.5-zein0.5 and SMN1.0-zein1.0 nanoparticles into air-dried BC films resulted in the increase of film thickness from 9.34 ± 2.56 μm to 22.79 ± 3.71 and 31.77 ± 0.83 μm. Thickness of freeze-dried BC films incorporated with SMN0.5-Zein0.5 and SMN1.0-Zein1.0 nanoparticles increased from 50.37 ± 2.77 μm to 106.00 ± 1.83 and 154.32 ± 7.93 μm, respectively. The thicker SMN1.0-Zein1.0/BC nanocomposite films represents the incorporation of more nanoparticles in the films, which can be correlated to their higher TPC (117.5 mg GAE/g in SMN1.0-Zein1.0/BC films vs. 69.4 mg GAE/g in SMN0.5-Zein0.5/BC films). Thus, SMN1.0-Zein1.0/BC nanocomposite films were selected for 13
further studies because of their higher TPC contents. Fig. 3C shows the XRD diffraction patterns of BC film and SMN1.0-Zein1.0/BC nanocomposite films. The peaks at 2θ angles of 15.1, 17.2, and 23.2º indicated that BC produced in this work were cellulose I (Vazquez, Foresti, Cerrutti, & Galvagno, 2013). However, the peak intensities of SMN1.0-Zein1.0/BC nanocomposite films at these 2θ angles were similar to that of the pure BC film, indicating that the interaction between SMN-Zein nanoparticles and BC fibers didn’t affect the crystallinity of BC fibers. Wetting properties of BC films and SMN1.0-Zein1.0/BC nanocomposite films were investigated by the contact angle of water droplet on the surface of the films. BC films have highly hydrophilic surfaces, so the water droplet angles of air-dried and freeze-dried BC films were small (28.14º and 24.25º). The contact angle values of the SMN1.0-Zein1.0/BC nanocomposite films increased to 41.50º and 31.62º, indicating that incorporation of SMN-Zein nanoparticles into the BC films increased the hydrophobicity of the film surfaces. Increase of hydrophobicity makes packaging films moisture resistant and provides an excellent barrier against water penetration, which may increase shelf life of food products. 3.4.Swelling and drug release of SMN-Zein/BC nanocomposite films The water uptakes of air-dried and freeze-dried SMN1.0-Zein1.0/BC composite films were measured. As shown in Fig. 4A, the air-dried and freeze-dried BC films reach equilibrium swelling after 1800 minutes. SMN-Zein/BC nanocomposite films demonstrated lower swelling degrees compared to original air-dried and freeze-dried BC films (60.2% and 152.7% vs. 269.8% and 306.9%) because the hydrophobic nature of
the SMN-Zein
nanoparticles that were incorporated in the BC films. There was no significant difference in swelling properties among the BC films incorporated with SMN0.5-Zein0.5 and SMN1.0-Zein1.0 nanoparticles (Fig. 4A). Silymarin is a hydrophobic crystalline compound that has very low solubility in water 14
(0.04 mg/mL). X-ray diffraction study shows that silymarin crystallinity decreased after formation of SMN-Zein nanoparticles (Fig. 4B). An active packaging film is designed for releasing antioxidant and antibacterial compound upon touching the food products in a controlled wet environment. Therefore, distilled water was used as a release medium in the drug release study. Fig. 4C shows that when silymarin powder was directly incorporated into BC films (SMN/BC films), silymarin was released from the films at a slow rate. Only 16.4% of the incprporated silymarin was released form the films after 120 h of release time. The slow release rate was due to the crystalline state and poor aqueous solubility of silymarin. The specific interaction between silymarine and zein results in the formation of well-dispersed SMN-Zein nanoparticles that have a relative larger surface area when compared to silymarin powder (Fig. 3C), thus is beneficial in dissolving silymarin. As shown in Fig. 4C, the air- and freeze-dried SMN-Zein/BC films released silymarin in a much higher ratio than the SMN/BC films, indicating that the nanocomposite films were effective in releasing the active component, silymarin, to improve the antioxidant and antibacterial functions of BC films. The SMN-Zein/BC films placed in the release medium for 24 h showed deformed BC matrix and SMN-Zein nanoparticles due to the release of incorporated silymarin (Fig. 4D). In the initial drug release stage (within 0 to 8 hours), the difference of the silymarin release profiles between SMN/BC films and SMN-Zein/BC films was due to the crystallinity and poor aqueous solubility of silymarin (Fig. 4C). Only a small portion of silymarin molecules that were well dispersed and were not aggregated in the BC films released from the SMN/BC films in the initial stage of drug release. However, after formation of SMN-Zein nanoparticles, the crystallinity of silymarin was decreased. Furthermore, the specific interaction between silymarine and zein results in the formation of well-dispersed SMN-Zein nanoparticles that have a relative larger surface area when compared to silymarin powder, 15
thus is beneficial in dissolving silymarin. The next stage of release showed a slow and sustained release of silymarin from SMN/BC film and SMN-Zein/BC films. Silymarin is a hydrophobic compound and is known to aggregate in water, causing a decreased release rate in the second stage of release. The slow release of silymarin from SMN-Zein/BC films in the second stage of release can be explained by the aggregation of SMN-Zein nanoparticles after immersing the composite films in water, as indicated in the TEM imaging (Fig. 4D). Besides, the freeze-dried SMN-Zein/BC films showed a slightly higher release rate in the second stage of release becaue freeze-drying is known to decrease bulk density of polymeric materials, including BC and zein. As shown in Fig. 4D, after 24 hours immersion in water (with shaking), the SMN-Zein nanoparticles still attached to the BC nanofibers (cross-section). Besides, more than 50% of incorporated silymarin were not released within 120 h of release (Fig. 4C). Silymarin is a flavonoid natural product extracted from the seeds of milk thistle. Silymarin can also be found in some foods and the most common food source is artichokes. Silymarin is generally considered safe and not toxic, and has very poor bioavailability due to its low water solubility. Some studies reported the treatment of various disease with high dosages (＞200 mg/kg/day) of silymarin without obvious side effect (Cetinkunar et al., 2015). In addition, when the films are used for food packaging, they just touch the food products but are not fully immersed in an aqueous solution, which will cause limited release of silymarin. Accordinglly, the composite films would not induce obvious negative effects due to excessive dosages. 3.5. Antioxidant and antibacterial test Previously, we have developed chitosan-based nanoparticles that have antioxidant and antibacterial activities (Yu et al., 2011; Tang et al., 2013). In this study, SMN-Zein nanoparticles were developed for the same purpose. Empty zein nanoparticles showed poor antioxidant property and the EC50 values against DPPH, ABTS·+ and superoxide anion were 16
897.5 ± 21.4 μg/ml, 55.3 ± 2.5 μg/ml and 3213.5 ± 165.7 μg/ml. Silymarin exhibited promising antioxidant potential against a variety of free radicals (Pientaweeratch, Panapisal, & Tansirikongkol, 2016). SMN-Zein nanoparticles with EC50 values of 38.5±1.1 μg/ml against DPPH,·2.4±0.3 μg/ml against ABTS·+, and 214.7±6.9 μg/ml against superoxide anion (Fig. 5A). The signal of the spin adduct (DMPO-OH) decreased by 48.1% under treatment by SMN-Zein nanoparticles (Fig. 5B), suggesting that the nanoparticles have a superior OH· scavenging activity comparable to free SMN (54.5% scavenging ratio). BC films didn't exhibit free radical scavenging properties against DPPH radical (Fig. 5C). However, the air-dried and freeze-dried SMN-Zein/BC nanocomposite films show effective free radicals scavenging capacity. Silymarin incorporated in the air-dried nanocomposite film was effective in scavenging DPPH free radicals for 72 h of antioxidant test. The antioxidant activity of the nanocomposite film could be maintained during long-term storage since antioxidant could be slowly released from the nanocomposite films (Fig. 4B). The free radical scavenging ratio of the freeze-dried nanocomposite film increased rapidly during 6 h and slowly increased thereafter, consequently, its free radical scavenging capacity was lower than that of the air-dried nanocomposite film (Fig. 5C). The antibacterial activity of the alkaline-treated BC films and SMN1.0-Zein1.0/BC nanocomposite films were shown in Fig. 5D. BC films didn’t show any antibacterial activity on the test bacteria (E. coli, S. aureus and P. aeruginosa). Bacterial populations of the nanocomposite films-treated bacterial cultures were significantly less compared to the alkaline-treated BC films-treated groups. The inhibition ratios of the air-dried nanocomposite films against E. coli, S. aureus and P. aeruginosa were 21.6 ± 2.8%, 62.1 ± 1.9%, 28.2 ± 4.9%, respectively. Silymarin has strong activity against Gram-positive bacteria but it is less effective against Gram-negative bacteria (Lee et al., 2003; Evren, & Yurtcu, 2015), therefore the nanocomposite films demonstrate better antibacterial efficacy against S. aureus than 17
against E. coli and P. aeruginosa. The freeze-dried nanocomposite film also shows an inhibitory ratio of 17.4 ± 3.6%, 53.5 ± 2.3% and 23.5 ± 5.4% against E. coli, S. aureus and P. aeruginosa (Fig. 5D), which are slightly less than that of the bacterial culture treated with air-dried nanocomposite film. The results suggested that BC films were endowed with antimicrobial properties after incorporating with SMN-Zein nanoparticles. 3.6. Prevention of spoilage and lipid peroxidation of salmon Carotenoids are known to be bleached under the conditions of deodorization that may involve exposure to oxidant species and blue-violet light irradiation (Yu et al., 2015). This bleaching process involves the cleavage of the double bonds and the formation of carbonyls or epoxides in the final products. SMN1.0-Zein1.0 nanoparticles showed 65% inhibition of oxidation in the β-carotene/linoleic acid/photoirradiation system (Fig. 6A), which is comparable to the inhibitory efficiency of RES1.0-Zein1.0 nanoparticles. Resveratrol (RES) is an effective antioxidant and the preparation of RES-Zein nanoparticles has been well studied (Joye et al., 2015). The results suggest that SMN1.0-Zein1.0 nanoparticles have an effective antioxidant activity that can protect β-carotene against linoleic acid peroxidation and photoirradiation-induced deodorization. Next, SMN1.0-Zein1.0 nanoparticles were incorporated into BC films and the effect of SMN1.0-Zein1.0/BC films packaging on the changes in TVNB values of salmon fillets during refrigerated storage were examined (Fig. 6B). The TVB-N values of the refrigerated salmon fillet in control group continuously increased (p < 0.05) from 7.65 mg N/100 g salmon to 66.6 mg N/100 g salmon fillet as the storage time increased to 16 days. When salmon fillets were packaged with SMN1.0-Zein1.0/BC nanocomposite films, the TVB-N contents were kept at lower levels for five days, compared with the control and the salmon fillets packaged with BC films. The TVB-N content of salmon packaged with air-dried SMN1.0-zein1.0/BC nanocomposite film was still below the TVB-N limit (30 mg N/100 g fish) after 12 days of 18
storage (Fig. 6B). Antibacterial and antiadherent/antibiofilm activity of silymarin against certain bacteria and fungi could reduce the production of volatile amines caused by microbial degradation of proteins (de Oliveira et al., 2015; Evren, & Yurtcu, 2015). The TBARS value of uncoated salmon muscle increased from 0.92 to 4.42 mg MDA/kg sample during after storage of 16 days due to lipid oxidation (Fig. 6C). The freeze-dried BC films didn’t significantly reduced lipid oxidation in salmon muscle compared to the uncoated salmon
(control
group).
However,
air-dried
and
freeze-dried
SMN1.0-Zein1.0/BC
nanocomposite films inhibited lipid peroxidation and the effects were significant (2.43 mg and 3.38 mg MDA/kg sample) compared to control (4.42 mg MDA/kg sample). Our previous study showed that caffeic acid-incorporated active films effectively inhibited lipid oxidation in fish oil emulsions (Yu et al., 2013). The protective effect of the SMN-Zein/BC films is attributed to the reason that silymarin can release from the films and exert a promising antioxidant potential against lipid oxidation (Pientaweeratch et al., 2016; Razavi-Azarkhiavi et al., 2014). Conclusion SMN-Zein/BC nanocomposite films were prepared by incorporation of SMN-Zein nanoparticles in BC nanofiber matrix. Antimicrobial and antioxidant activity of
BC films
were enhanced with the incorporation of bioactive SMN-Zein nanoparticles. The results demonstrated the efficacy the air-dried SMN-Zein/BC nanocomposite film in preventing the deterioration of salmon muscle and slowing down the lipid oxidation, thus allowing the packaged fish with extended shelf life in cold storage. Acknowledgments The authors gratefully acknowledge the financial support provided by the National Science Council, Taiwan, Republic of China (NSC 101-2221-E-038-016-MY3 and MOST 103-2313-B-019-003-MY3). 19
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Figure captions Fig. 1. Characterization of native and alkaline-treated BC films: (A) SEM micrograph, (B) XRD pattern, (C) FTIR spectra, (D) TGA and DTG curves . Fig. 2. (A) Influence of pH on the mean particle size and zeta potential of SMN1.0-Zein1.0 nanoparticles, (B) TEM micrographs of SMN1.0-Zein1.0 (a) and SMN0.5-Zein0.5 (b) nanoparticles, (C) fluorescence spectra of zein and SMN-Zein nanoparticles, (D) FTIR spectra of zein, SMN and the prepared SMN-Zein nanoparticles. Fig. 3. Characterization of SMN-Zein/BC nanocomposite films: (A) SEM micrograph, (B) 24
FT-IR spectra, (C) XRD pattern, (D) water drop contact angle. Fig. 4. (A) Swelling properties of BC films and SMN1.0-Zein1.0/BC nanocomposite films, (B) XRD pattern of SMN powder and crystal, and SMN-Zein nanoparticles, (C) silymarin release profiles from SMN1.0-Zein1.0/BC nanocomposite films, (D) SEM micrographs of SMN1.0-Zein1.0/BC nanocomposite films after 24 h placed in release medium. Fig. 5. Antioxidant and antibacterial properties of SMN1.0-Zein1.0/BC nanocomposite films: (A) EC50 of DPPH free radical scavenging activity, (B) EPR spectra of DMPO spin adducts of hydroxyl radical, (C) the continuous DPPH free radical scavenging behavior, (D) inhibition ratio of the nanocomposite films against E. coli, S. aureus and P. aeruginosa Fig. 6 Antioxidant and antibacterial properties of SMN1.0-Zein1.0/BC nanocomposite films: (A) EC50 of DPPH free radical scavenging activity, (B) EPR spectra of DMPO spin adducts of hydroxyl radical, (C) the continuous DPPH free radical scavenging behavior, (D) inhibition ratio of the nanocomposite films against E. coli, S. aureus and P. aeruginosa
25
26
27
28
29
30
31
Table 1. Mean particle sizes, zeta potentials and SMN loading efficiency of SMN-Zein nanoparticles. silymarin:zein
mean particle size
zeta potential
loading efficiency
(wt %/wt %)
(nm)
(mV)
(%)
0.50:0.20
2898.5 ± 38.3
–
–
0.50:0.30
767.5 ± 15.6
25.4 ± 0.3
61.5 ± 4.2
0.50:0.40
228.7 ± 6.8
23.9 ± 0.6
68.8 ± 5.1
Siymarin 0.5 wt%
32
0.50:0.50
198.5 ± 9.8
19.7 ± 0.8
73.4 ± 3.8
1.00:0.70
1690.5 ± 45.2
–
–
1.00:0.80
526.7 ± 3.8
20.5 ± 0.7
85.5 ± 5.7
1.00:0.90
229.8 ± 4.7
17.7 ± 0.5
87.2 ± 3.9
1.00:1.00
214.4 ± 1.4
14.9 ± 0.6
94.3 ± 2.3
Siymarin 1.0 wt%
33